Polymers for reversing heparin-based anticoagulation

ABSTRACT

Embodiments presented herein relate to various polymers. Some of the polymer embodiments are heparin binding polymers. Some embodiments of the heparin binding polymers can be employed to bind to heparin for methods such as separating, purifying, removing, and/or isolating heparin and heparin like molecules.

RELATED APPLICATIONS

This application is a divisional under 35 U.S.C §121 of U.S. applicationSer. No. 13/458,899, filed Apr. 27, 2012, now U.S. Pat. No. 8,637,008,which is a continuation under 35 U.S.C §120 of U.S. application Ser. No.13/504,841, filed Apr. 27, 2012, now U.S. Pat. No. 8,519,189, which wasthe U.S. National Phase entry under 35 U.S.C. §371 of InternationalApplication PCT/CA2011/050603, filed Sep. 27, 2011, entitled “POLYMERSFOR REVERSING HEPARIN-BASED ANTICOAGULATION,” which claims priority toU.S. Provisional Application Ser. No. 61/492,299, filed Jun. 1, 2011,the entireties of which are incorporated herein by reference.

TECHNICAL FIELD

The present embodiments relates to novel polymers that bind to heparin,heparin derivatives and heparinoids and methods for making suchpolymers.

BACKGROUND

Heparin is an anionic compound involved in a variety of biologicalprocesses including blood coagulation. Heparin derivatives used incurrent clinical anticoagulation therapy include unfractionated heparin(UFH), low molecular weight heparin (LMWH), ultra low molecular weightheparin (ULMWH) and the synthetic pentasccharide derivativesfondaparunix and idraparinux.

Heparinoids can be naturally occurring and synthetic highly-sulfatedpolysaccharides of similar structure to heparin. Heparinoid preparationshave been used for a wide range of applications including asanticoagulant and anti-inflammatories and they have been claimed to havehypolipidemic properties.

Heparin neutralization, which can be desired when a subject is given toomuch heparin, can be achieved, for example, via protamine or throughfiltration of the blood through an extracorporeal device.

SUMMARY

Some embodiments provided herein relate to heparin binding polymers. Insome embodiments, the polymer has a first dendritic polyol and one ormore cationic moieties attached to the first dendritic polyol.

In some embodiments, a heparin binding device is provided. The devicecan have a support and a heparin binding polymer immobilized on thesupport. The heparin binding polymer can have a first dendritic polyoland one or more cationic moieties attached to the first dendriticpolyol.

In some embodiments, a method of making a heparin binding polymer isprovided. The method can include combining a glycidol and a PEG-epoxide,methylating the glycidol and the PEG-epoxide to form a hyperbranchedpolyglycerol, tosylating the hyperbranched polyglycerol to form atosylated hyperbranched polyglycerol, and coupling the tosylatedhyperbranched polyglycerol with at least one amine group, thereby makinga heparin binding polymer.

In some embodiments, a method of counteracting heparin in a subject isprovided. The method can include administering a heparin binding polymerto a subject. The heparin binding polymer can include a first dendriticpolyol and one or more cationic moieties attached to the first dendriticpolyol. The heparin binding protein binds to heparin and therebycounteracts heparin in the subject.

In some embodiments, a method of processing a subject's blood isprovided. The method can include providing a heparin binding device. Thedevice can include a support and a heparin binding polymer immobilizedon the support. The heparin binding polymer can include a firstdendritic polyol and one or more cationic moieties attached to the firstdendritic polyol. The method can further include withdrawing blood froma subject, contacting the blood to the heparin binding polymer, andreturning at least part of the blood to the subject, thereby processingthe subject's blood.

In some embodiments, a method of concentrating heparin is provided. Themethod can include providing a support and a heparin binding polymerimmobilized on the support. The heparin binding polymer can include afirst dendritic polyol and one or more cationic moieties attached to thefirst dendritic polyol. The method can further include contacting afirst fluid including heparin with the heparin binding polymer andflowing the fluid off of the heparin binding polymer. This can therebyconcentrate heparin.

In some embodiments, a heparin binding composition is provided. Thecomposition can include a first heparin binding polymer having a firstdendritic polyol and one or more cationic moieties attached to the firstdendritic polyol. The composition can further include a pharmaceuticallyacceptable carrier.

In some embodiments, a heparin binding macromolecule is provided. Themacromolecule can include a hyperbranched polyglycidol core, at leastone polyvalent cation attached to the hyperbranched polyglycidol core,and at least one protective moiety attached to the hyperbranchedpolyglycidol core.

In some embodiments, a heparin composition is provided. The compositioncan include heparin and a heparin binding polymer. The heparin bindingpolymer can include a first dendritic polyol and one or more cationicmoieties attached to the first dendritic polyol.

In some embodiments, a kit is provided. The kit can include a firstcontainer including heparin and a second container including a heparinbinding polymer. The heparin binding polymer can include a firstdendritic polyol and one or more cationic moieties attached to the firstdendritic polyol.

In some embodiments, the heparin binding polymer can be used for bindingand drug delivery of anionic drug molecules such as heparins, syntheticpentasaccharide anticoagulants, low and ultra low molecular weightheparins or methotrexate or similar compounds. The foregoing summary isillustrative only and is not intended to be in any way limiting. Inaddition to the illustrative aspects, embodiments, and featuresdescribed above, further aspects, embodiments, and features will becomeapparent by reference to the drawings and the following detaileddescription.

In some embodiments, a controlled release delivery device is provided.The device can include a surface and a heparin binding polymer attachedto the surface. The heparin binding polymer can include a firstdendritic polyol and one or more cationic moieties attached to the firstdendritic polyol. The controlled release delivery device can alsoinclude heparin. In some embodiments, the heparin is bound to theheparin binding polymer.

In some embodiments, a controlled release heparin composition isprovided. The composition can include a heparin binding polymer. Theheparin binding polymer can include a first dendritic polyol and one ormore cationic moieties attached to the first dendritic polyol. Thecontrolled release heparin composition can also include heparin. In someembodiments, the heparin is bound to the heparin binding polymer.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A depicts a schematic representation of some embodiments of aheparin binding synthetic polyvalent cationic macromolecule withdifferent amine groups (R=tetra-amine (4 nitrogens), hexa-amine (6nitrogens) and deca-amine (10 nitrogens).

FIG. 1B depicts various forms of heparin.

FIG. 2A is a depiction of some embodiments for the synthesis of heparinand heparin-derivative binding polymers with high charge density.

FIG. 2B is a depiction of some embodiments of the synthesis of heparinand heparin-derivative binding polymer with high charge density. Theembodiment employs MPEG-400 and an amine with four nitrogen atoms.

FIG. 3 is a depiction of an embodiment of the design of heparin bindingsynthetic polyvalent cationic macromolecule (HBSPCM).

FIGS. 4A and 4B are graphs demonstrating heparin binding syntheticpolyvalent cationic macromolecule JNK-1 neutralization of A)unfractionated heparin (UFH) B) LMWH (Tinzaparin) in human blood underin vitro conditions. Protamine neutralization is given as a comparison.

FIGS. 5A-5D are graphs depicting the percentage of unfractionatedheparin (UFH) and low molecular weight heparin (LMWH) neutralizationcalculated from an activated partial thromboplastin time (APTT) assay byheparin binding synthetic polyvalent cationic macromolecule JNK-1 andprotamine.

FIGS. 6A-6D are graphs depicting heparin binding synthetic polyvalentcationic macromolecule JNK-1 neutralization of unfractionated heparin(UFH) and low molecular weight heparin (LMWH) Tinzaparin using athromboelastograph (TEG) as measured by a thromboelastograph in humanwhole blood.

FIGS. 7A-7B are graphs depicting heparin binding synthetic polyvalentcationic macromolecule JNK-1 neutralization of unfractionated heparin(UFH) and low molecular weight heparin (LMWH) Tinzaparin usingthromboelastograph (TEG) in terms of coagulation time and clot strengthin human whole blood.

FIGS. 8A-8C are graphs depicting heparin binding synthetic polyvalentcationic macromolecule JNK-1 neutralization of unfractionated heparin(UFH) and low molecular weight heparin (LMWH) Tinzaparin in rats.

FIG. 8D is a graph depicting in vivo UFH and LMWH (by HBSPCM-3)neutralization in rats.

FIG. 9 is a graph depicting electrostatic charge neutralization ofunfractionated heparin (UFH) and low molecular weight heparin (LMWH) byheparin binding synthetic polyvalent cationic macromolecule JNK-1.

FIG. 10A is a bar graph depicting complement activation upon interactionof heparin binding synthetic polyvalent cationic macromolecule JNK-1with platelet poor plasma.

FIG. 10B is a bar graph depicting platelet activation upon interactionof JNK-1 with platelet rich plasma at 37 C.

FIGS. 11A and 11B are bar graphs depicting the effect of heparin bindingsynthetic polyvalent cationic macromolecule JNK-1 on prothrombin time(PT) and activated partial thromboplastin time (APTT) in platelet poorplasma (PPP).

FIGS. 12A-12D depict optical micrographs of human red blood cells after1 hour incubation with heparin binding synthetic polyvalent cationicmacromolecule JNK-1 at different concentrations in whole blood at 37° C.

FIG. 13 is a synthetic scheme for disulfide-containing heparin bindingsynthetic polyvalent cationic macromolecule (HBSPCM).

FIG. 14 is a depiction of a structure of a ketal group-containingheparin binding synthetic polyvalent cationic macromolecule (HBSPCM).

FIG. 15 is a bar graph depicting a blood compatibility analysis(activated partial thromboplastin time (APTT) biocompatibility) ofHBSPCM polymers JNK-3 through JNK-8-control as compared to protamine andsaline.

FIG. 16 is a graph depicting blood compatibility analysis usingthromboelastograph (TEG) in terms of coagulation time and kineticsparameter of HBSPCM polymers JNK-3 through JNK-8-control as compared toprotamine and saline.

FIGS. 17A and 17B are photos and bar charts depicting red blood celllysis after incubation with HBSPCM polymers JNK-3 through JNK-8 ascompared to protamine and saline.

FIGS. 18A-18C are graphs depicting the neutralization of unfractionatedheparin (FIG. 18A), Tinzaparin (FIG. 18B) and Enoxaparin (FIG. 18C) byHBSPCM polymers JNK-1 through JNK-8 as compared to protamine in humanblood in vitro.

FIG. 10 is a graph depicting the neutralization of low molecular weightheparin using a thromboelastograph (TEG) in human whole blood by HBSPCMpolymers JNK-5 and JNK-6.

FIG. 20 is a bar chart depicting complement activation by the HBSPCMpolymers JNK-3 to JNK-8 in comparison to protamine and saline.

FIGS. 21A and 21B are graphs depicting the ability of HBSPCM polymerJNK-3 to neutralize unfractionated heparin (FIG. 21A) and Enoxaparin(FIG. 21B) in living rats.

FIG. 22 is a graph plotting body weight over time (0 to 29 days postinjection) of female BALB/c mice treated with HBSPCM polymer JNK-3 bybolus intravenous injection.

FIG. 23 is a graph depicting LDH activity in serum of female BALB/c micetreated with HBSPCM polymer JNK-3 after a bolus intravenous injection.

FIGS. 24A-24C are graphs depicting neutralization of unfractionatedheparin (UFH) (FIG. 24A), Tinzaparin (FIG. 24B) and Enoxaparin (FIG.24C) by HBSPCM polymers JNK-9, JNK-10 and JNK-11 as compared toprotamine in human plasma in vitro.

FIG. 25 is a bar graph depicting blood compatibility (complementactivation) of HBSPCM and the effect of the number of charges on thepolymer.

FIG. 26 is a bar graph depicting blood compatibility (plateletactivation) of HBSPCM and the effect of the number of charges on thepolymer.

FIG. 27 is a graph depicting the tolerance of mice to HBSPCM (JNK-1)

FIG. 28 is a graph depicting the tolerance of mice to protamine.

FIG. 29A and FIG. 29B are graphs depicting the bio-distribution andpharmacokinetics of HBSPCM-3 in BALB/c mice.

FIGS. 30A, 30B, 30C, and 30D are graphs depicting the bio-distributionand pharmacokinetics of HBSPCM-3 in BALB/c mice.

FIGS. 31A, 31B, 31C, and 31D are graphs depicting the bio-distributionand pharmacokinetics of HBSPCM-1 in BALB/c mice.

FIGS. 32A and 32B are graphs depicting the bio-distribution andpharmacokinetics of HBSPCM-1 in BALB/c mice.

DETAILED DESCRIPTION

A complication and health concern associated with the use of heparin andits derivatives for anticoagulation therapy is bleeding. Antidotes areneeded to reverse the effects of heparin or its derivatives to avoidbleeding complications while maintaining sufficient levels of theanticoagulant to prevent thrombosis. Apart from bleeding complications,neutralization of anticoagulant drugs following major surgicalprocedures can be useful to prevent postoperative complications.

The only antidote available for UFH approved by the U.S. Food and DrugAdministration (FDA) is protamine. There are numerous disadvantages tousing protamine as a heparin antidote, including its narrow window fortherapeutic dosage, poorer outcomes for blood transfusions and the lackof a reliable method for the quantification of protamine in blood.Protamine is associated with a number of anaphylactic adverse drugreactions (Horrow et al. 1985 Anesth Analg. 64:348-61; Kimmel et al.1998 J Clin Epidemiol. 51:1-10).

Biospecific polybases, including polypeptides, synthetic peptides andcationic polymers, can bind and neutralize heparin and heparinderivatives to overcome the disadvantages of protamine and otherantidotes. As disclosed herein, the efficiency of neutralization ofheparin and heparin derivatives depends on the net charge, chargedensity and molecular structure of polybases.

Provided herein are various molecules and compositions that can be usedfor binding to heparin. It has been discovered that a class ofbiocompatible hyperbranched polyol polymers have the ability to bind toheparin and heparin derivatives and, in some embodiments, to act as anantidote to heparin-based anticoagulation therapy. In some embodiments,the molecules are hyperbranched polyether polyol polymers. It isunderstood by the present inventors that this new use is distinct fromother uses of hyperbranched polyether polyols, including their use ascarriers, drug delivery vehicles or modulating an energy substrate. Insome embodiments, the molecules include a dendritic polyol attached toone or more cationic moieties. As described in greater detail below, insome embodiments, such molecules can be employed to purify and/orisolate heparin, among other uses.

In the following detailed description, reference is made to theaccompanying drawings, which form a part hereof. In the drawings,similar symbols typically identify similar components, unless contextdictates otherwise. The illustrative embodiments described in thedetailed description, drawings, and claims are not meant to be limiting.Other embodiments may be utilized, and other changes may be made,without departing from the spirit or scope of the subject matterpresented herein. It will be readily understood that the aspects of thepresent disclosure, as generally described herein, and illustrated inthe Figures, can be arranged, substituted, combined, separated, anddesigned in a wide variety of different configurations, all of which areexplicitly contemplated herein.

The present disclosure provides a general set of definitions (which caninclude various embodiments) of terms that are relevant to some of theembodiments. The disclosure then provides a detailed set of embodimentsand examples. While various sections of the disclosure include headings,these headings are not to be interpreted in a limiting manner, and arepresent merely for the sake of convenience.

DEFINITIONS AND EMBODIMENTS

Any terms not directly defined herein shall be understood to have allthe meanings commonly associated with them as understood within the art.Certain terms are discussed below, or elsewhere in the specification, toprovide additional guidance to the practitioner in describing thedevices, methods and the like of various embodiments, and how to make oruse them. It will be appreciated that the same thing may be said in morethan one way. Consequently, alternative language and synonyms may beused for any one or more of the terms discussed herein. No significanceis to be placed upon whether or not a term is elaborated or discussedherein. Some synonyms or substitutable methods, materials and the likeare provided. Recital of one or a few synonyms or equivalents does notexclude use of other synonyms or equivalents, unless it is explicitlystated. Use of examples in the specification, including examples ofterms, is for illustrative purposes only and does not limit the scopeand meaning of the embodiments herein.

As used herein, a “HPG polymer” refers to a hyperbranched polyetherpolyol, including hyperbranched polyglycerol (HPG) and heparin-bindingsynthetic polyvalent cationic macromolecules (HBSPCMs). Various methodsfor synthesizing and modifying HPG polymers are described herein, and inthe literature. Initiating species can include polyols and polyamine. Insome embodiments, HPG polymers can be further derivatized with alkylgroups, polyethylene glycol groups, amine groups, sulfate groups,carbohydrates, peptides, amino acids and the like.

As used herein, a “HBSPCM polymer” or “HBSPCM” refers to aheparin-binding synthetic polyvalent cationic macromolecule. Variousmethods for synthesizing and modifying HBSPCM polymers are describedherein. In some embodiments, the core of the HBSPCM polymer can includea HPG polymer. The core of the HBSPCM can be capped with short chainpolyethylene glycols (PEGs) to create a macromolecule.

The term “heparin binding polymer” is generic to both “HPG polymer” and“HBSPCM polymer”.

“Subject”, as used herein, refers to an entity to receive the molecule,compound, treatment, etc. In some embodiments, the subject is a humanpatient, human test subject, a primate, or other mammal, such as a rat,mouse, dog, cat, cow, pig, sheep, monkey or the like.

As used herein, a “pharmaceutically acceptable excipient” includes anyand all solvents, dispersion media, coatings, antibacterial,antimicrobial or antifungal agents, isotonic and absorption delayingagents, and the like that are physiologically compatible. The excipientmay be suitable for intravenous or intraarterial administration. Theexcipient can include sterile aqueous solutions or dispersions forextemporaneous preparation of sterile injectable solutions ordispersion. Examples of sterile aqueous solutions include saline,Ringer's lactate or other solutions as may be known in the art. Use ofsuch media in the preparation of a medicament is known in the art.

As used herein, a “pharmacologically effective amount” of a medicamentrefers to using an amount of a medicament present in such aconcentration to result in a therapeutic level of drug delivered overthe term that the drug is used. This can be dependent on mode ofdelivery, time period of the dosage, age, weight, general health, sexand diet of the subject receiving the medicament.

In some embodiments, HPG polymers can be administered to a subject toneutralize the effect of heparin or heparin derivatives on the subject.As used herein, the term “neutralize” includes, in its variousgrammatical forms (e.g., “neutralized”, “neutralization”,“neutralizing”, etc.), binding to in any capacity, immobilize,counteract, change, alter, modify.

The term “heparin” as used herein is generic to both traditional,naturally occurring forms of heparin, as well as heparin derivativesand/or artificial forms of heparin. Heparin is a glycosaminoglycan andacts as an anticoagulant. The molecule has a negative charge density.Some forms of heparin have average molecular weights from 2 kDa to 30kDa, such as between 12 kDa and 15 kDa. FIG. 1B depicts some embodimentsof heparin. Low molecular weight embodiments of heparin includeenoxaparin, dalteparin, fondaparinux, tinzaparin and ultra low molecularweight embodiments of heparin include semuloparin. Heparin sulfate isincluded within the genus of the term “heparin”.

LMWH is enzymatically or chemically modified UFH with highbioavailability, predictable pharmacokinetics and dose-response andtherefore has a lower risk of bleeding complications. Fondaparinux andidraparinux are synthetic versions of the pentasaccharide moietyresponsible for anti-factor Xa activity in UFH and LMWH. However, theanticoagulant activity of LMWHs, fondaparinux and idraparinux cannot befully neutralized with a currently available antidote (Makris et al.2000 Br J Haematol. 108:884-5). Ultra low molecular weight heparin suchas semuloparin has high anti-factor Xa and residual anti-factor II aactivities and is currently under development for treatment of venousthromboembolism (VTE) and Cancer therapy.

The term “polyol” as used herein denotes an alcohol including two ormore hydroxyl groups. This includes diols, triols, tetrols, etc.

The term “dendritic” when used in regard to polymers denotes abranching, tree-like structure (see, e.g., FIG. 1A or FIG. 2A). In someembodiments, dendritic polymers can have a large number of endfunctional groups and have low melt and solution viscosities.

The term “cationic moiety” denotes a chemical moiety that has a positivecharge.

The term “protective moiety” denotes a chemical moiety that can assistin reducing non-specific interactions between a polyglycerol core and/orpolyvalent cationic groups and chemicals other than heparin. Examplesinclude, for example, polyethylene glycol.

The term “polyethylene glycol” or “PEG” denotes an oligomer and/orpolymer of ethylene oxide and encompasses, for example, polyethyleneoxide (PEO) and polyoxyethylene (POE).

The term “polyether polyol” denotes a polyol that has the etherfunctional group in the chain.

The term “hyperbranched” in reference to polymers denotes a polymerhaving 3 or more chain ends. In some embodiments, hyperbranched polymersare not perfectly branched. In some embodiments, hyperbranched polymerscan be prepared in a one-step procedure. In some embodiments,hyperbranched polymers are molecular constructs having a branchedstructure, generally around a core. In some embodiments, their structuregenerally lacks symmetry, the base units or monomers used to constructthe hyperbranched polymer can be of diverse nature and theirdistribution is non-uniform. In some embodiments, the branches of thepolymer can be of different natures and lengths. In some embodiments,the number of base units, or monomers, can be different depending on thedifferent branching. In some embodiments, while at the same time beingasymmetrical, hyperbranched polymers can have: a branched structure,around a core, successive generations or layers of branching, and alayer of end chains. In some embodiments, the chain ends can beconnected to cationic moieties and/or protective moieties. In someembodiments, there are three to 100,000 chain ends, e.g., 3-50,000,3-20,000, 3-10,000, 3-5,000, or 100-1000.

The term “cleavage site” denotes a bond that can be selectively cleavedover the remaining section of the polymer backbone. Cleavage sitesinclude, for example, disulfide bonds and ketal groups (see, e.g., FIG.13 and FIG. 14).

The term “ketal” group denotes a compound that includes the generalFormula: Ia:

R¹ and R² can be an alkyl or aryl.

An “amine” denotes an organic compound and/or functional group thatinclude a basic nitrogen with a lone pair.

The term “alkyl,” as used herein, means any unbranched or branched,substituted or unsubstituted, saturated hydrocarbon, with C₁-C₁₂unbranched, saturated, unsubstituted hydrocarbons, including, e.g.,methyl, ethyl, isobutyl, and tert-butylpropyl, and pentyl.

The term “alkoxy” refers to any unbranched, or branched, substituted orunsubstituted, saturated or unsaturated ether, including C₁-C₆unbranched, saturated, unsubstituted ethers. Dimethyl, diethyl,methyl-isobutyl, and methyl-tert-butyl ethers also included. The term“cycloalkoxy” refers to any non-aromatic hydrocarbon ring, includingthose having five to twelve atoms in the ring.

The “number average molecular weight” or “Mn” is the ordinary arithmeticmean or average of the molecular weights of the individualmacromolecules. It can be determined by measuring the molecular weightof n polymer molecules, summing the weights, and dividing by n.

The phrase “degree of pegylation” denotes the fraction of the totalend-functional (hydroxyl) groups of the macromolecule that has beenmodified with polyethylene glycol (PEG).

A glycidol is an organic compound that includes both an epoxide andalcohol functional groups linked by a methylene (CH₂) group or (CH₂)_(n)where n=2-10.

The term “hydrodynamic radius” denotes a theoretical hydrodynamic radiusR_(hyd). R_(hyd) is defined by:

$\frac{1}{R_{hyd}}\overset{def}{=}{\frac{1}{N^{2}}\left\langle {\sum\limits_{i \neq j}\frac{1}{r_{ij}}} \right\rangle}$

where r_(ij) is the distance between subparticles i and j, and where theangular brackets

. . .

represent an ensemble average.

General Embodiments Heparin Binding Molecules

In some embodiments, the heparin binding molecule is a heparin bindingpolymer. In some embodiments, the heparin binding polymer can include afirst dendritic polyol and one or more cationic moieties attached to thefirst dendritic polyol. In some embodiments the heparin binding polymercan include a first linear polyol and one or more cationic moietiesattached to the first linear polyol.

In some embodiments, the heparin binding polymer can further include aprotective moiety covalently attached to the first dendritic polyol. Insome embodiments, the protective moiety includes polyethylene glycol(e.g., as shown in FIG. 1A). In some embodiments, a new layer ofpolyglycerol can be a protective moiety. In some embodiments, theprotective moiety can be a biocompatible polymer such as polyvinylpyrolidone, carbohydrate polymers, polypeptides, linear polyglycerol,polymers and copolymers of PEG acrylates and methacrylates, PEGacrylamide and methacrylamides.

In some embodiments, the heparin binding polymer is a polyether polyol.In some embodiments, the heparin binding polymer is a carbohydratepolymers, linear polyglycerol, polypeptides, branched or linear polymersand copolymers of PEG acrylates and methacrylates, PEG acrylamide andmethacrylamides.

In some embodiments, the polyether polyol can be hyperbranched. In someembodiments, the degree of branching in the range of 0.0 to 0.95, forexample, in the range of 0.0, 0.05, 0.1 to 0.9, 0.2-0.7, 0.4-0.65, orabout 0.5. In some embodiments, the heparin binding polymer is ahyperbranched polyglycerol or a linear polyglycerol.

In some embodiments, the heparin binding polymer can include a polyetherpolyol, which can be attached to a support. In some embodiments, theheparin binding polymer is attached to the support via a disulfide bond.In some embodiments, the polymer is attached to the support via anacetal bond or a ketal bond. The support can be any material to whichthe molecule can be attached. In some embodiments, the support can be abead or a flat surface.

In some embodiments, the heparin binding polymer includes a core unitthat includes a C₁₋₁₈ alkyl substituted with two or more of —OR¹. Insome embodiments, each R¹ is independently selected from at least oneof: a hydrogen, a cationic moiety, and a polymer segment having monomerunits represented by Formula (III):

n can be an integer from 1 to 10,000. Each R² can be independentlyselected from: a hydrogen, carbon, a cationic moiety, R¹, and a polymersegment represented by Formula (IV):

m can be an integer from 1 to 10,000.

Each R³ can be independently selected from: an oxygen cationic moiety, ahydroxyl, and a polymer segment represented by Formula (IV). m can be aninteger from 1 to 10,000 and each R⁴ can be a C₁₋₆ alkoxy.

In some embodiments, the core unit is represented by Formula (V):

In some embodiments, the cationic moiety includes Formula (VI):

wherein the heparin binding polymer includes an average of 4 to 250cationic moieties. In some embodiments, the heparin binding polymerincludes an average of 1, 2, 4, 5, 16, 20, 24, 23, 8, 11, 16, or 36cationic moieties. In some embodiments, the polymer segment can berepresented by Formula (IV):

and includes a polyethylene glycol having a size of 400 Da.

In some embodiments, the number average molecular weight (Mn) of thepolymer segment represented by Formula (IV) is about 400 Da. In someembodiments, the average molecular weight (Mn) of the polymer segmentrepresented by Formula (IV) is between 50 and 40,000 Da, for example 100to 800, 200 to 700, 300 to 600, 300 to 500, or 350 to 450.

In some embodiments, the heparin binding polymer has a degree ofPEGylation that is from about 5% to about 95%. In some embodiments, thepolymer has a degree of PEGylation that is from about 20% to about 40%.

In some embodiments, the cationic moiety is selected from at least oneof the group of: Formula (VI), Formula (VII), and Formula (VIII)

In some embodiments, the heparin binding polymer is at least one of:JNK-4, JNK-5, JNK-6, JNK-3, JNK-7, JNK-8, JNK-9, JNK-10, JNK-11, and anycombination thereof.

In some embodiments, the heparin binding molecule is a hyperbranchedpolyether polyol, which can be a hyperbranched polyglycerol (HPG).

In some embodiments, the hyperbranched polyether polyol polymerstructure is a synthetic polycationic dendritic polymer based on HPGcapped with short chain polyethylene glycols (PEGs) to create amacromolecule.

In some embodiments, the hyperbranched polyether polyol can furtherinclude one or more tetra (4 nitrogens) amine groups. In someembodiments, the hyperbranched polyether polyol can include one or morehexa (6 nitrogens), octa (8 nitrogens), or deca (10 nitrogens) aminegroups.

In some embodiments, the hyperbranched polyether polyol can be ofvarying molecular weights of varying charge to optimize the reversal ofanticoagulation due to a specific heparin species, such as low molecularweight heparin, or to otherwise optimize the administration of thepolymer to a subject.

In some embodiments, the heparin binding polymer is the polymer shown inFIG. 1A. In some embodiments, the heparin binding polymer shown in FIG.1A lacks the PEG groups in FIG. 1A (and thus would not be a“macromolecule” as the term is used herein). In some embodiments, onecan employ a polymer that lacks PEG for heparin neutralization. In someembodiments, the heparin binding polymer is part of a macromolecule (asshown in FIG. 1A and FIG. 3).

In some embodiments, the heparin binding polymer has one or more Rgroups that includes 4, 6, 8, or 10 nitrogens. In some embodiments, eachR group of the heparin binding polymer has 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25, 30 or morenitrogens, including any range defined between any two of the precedingvalues and any range greater than any one of the preceding values.

In some embodiments, all of the R groups for a particular heparinbinding polymer are the same. In some embodiments, a single heparinbinding polymer can have different types and/or sizes of R groups. Insome embodiments, a single heparin binding polymer can have two or moreof 4N, 6N, 8N, and 10N R groups. In some embodiments, the R groups arecationic groups. In some embodiments, the spacer between the nitrogenatoms in the R groups can be (CH₂)_(n) where n=1 to 10 (e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, or 10) or the spacer can have a cyclic aliphaticstructure.

In some embodiments, heparin binding polymers and/or hyperbranchedpolyglycidols (HPGs) can be synthesized using various methods known inthe art (Kainthan et al. 2006 Biomacromolecules. 7(3):703-709; Kainthanet al. 2007 Biomaterials. 28(32): 4779-4787). In some embodiments, theheparin binding polymers and/or hyperbranched polyglycidol polymer is aheparin-binding synthetic polyvalent cationic macromolecule (HBSPCM),e.g., as shown in FIG. 1A. In such embodiments, the HBSPCM canincorporate multiple high binding cationic units (R) attached on adendritic HPG core, and can be capped with short chain polyethyleneglycols (PEGs) to generate a polyvalent cationic macromolecule, as shownin FIG. 1A. In some embodiments, the cationic unit (R) is a methylatedtetramine with four cationic amine groups per cationic units (R=4N) andwill produce high binding affinity for heparin and heparin derivatives.FIG. 3 is a graphical interpretation of one embodiment with a HPG coreon which polyvalent cationic groups are attached (C) and capped with aprotective barrier to non-specific interactions. In some embodiments,the protective barrier is composed of short chain PEGs of approximately400 Da each. The presence of a protective layer of short chain PEGs onthe HBSPCM makes it highly biocompatible.

In some embodiments, the hydroxyl groups on the dendritic HPG core willbe used for adding cationic units to the HBSPCM. The number of hydroxylgroup is equal to degree of polymerization; e.g., each 74 Da molecularweight will have one hydroxyl group. The design of HBSPCM enablesmultiple embodiments including, but not limited to, changing the numberof cationic units, changing the size of the HPG core (molecular weight)while keeping the PEG chain length constant, and changing the number ofPEG chains attached to HPG core.

In some embodiments, the HPG core capped with PEGs is modified in acontrolled manner by the covalent attachment of multi-valent cationicgroups through a three step synthetic method involving tosylation ofhydroxyl groups, coupling with amines (or other cationic groups), andmethylation as shown in FIGS. 2A and/or 2B. Possible amine groupsinclude, but are not limited to, tetra-amine, hexa-amine, octa-amine,deca-amine groups, and any other amine groups disclosed herein. TheHBSPCM properties such as, but not limited to, molecular weight,molecular weight distribution, PEG content, charge density and plasmaresidence time can be controlled by methods known in the literature,such as, but not limited to, the anionic ring opening polymerization ofglycidol and MPEG-epoxide and similar methods (Kainthan et al. 2006Biomacromolecules. 7(3):703-709; Nair et al. 2007 Prog Polymer Sci. 32(8-9):762-798; Nisha et al. 2004 Langmuir. 20(6):2386-2396; Nisha et al.2004 Langmuir. 20(20):8468-8475; Kizhakkedathu et al. 2005 Macromol.Biosci. 5(6):549-558).

Examples of synthesized HBSPCMs are shown in Tables 1, 2, and 3. Twoembodiments, JNK-1 (116 000 Da) and JNK-2 (48 000 Da), have 69 and 28cationic groups (R; 4 charges/cationic group), respectively. Thehydrodynamic radii of these molecules are in the range of 4 to 10 nmwith a PEG content of approximately 75% by weight.

In some embodiments, the interactions between cationic and anioniccharges are maximized due to the dendritic nature of the HPG core andPEG caps (maximum surface exposure of charges and multivalent effect).This characteristic has advantages over linear polymers, which cannotmaximize surface exposure of charges.

In some embodiments of the technology, biodegradable HBSPCMs aresynthesized for optimal clearance of HBSPCM and its complexes from thevascular system and from the body. These HBSPCMs will have precisedegradation points. Degradation points can be constructed from one ormore elements from the following categories including, but not limitedto, disulphide bonds (FIG. 13) and ketal groups (FIG. 14). In oneembodiment, the disulfide initiator is prepared by reaction ofocta-amine and 2 hydroxy, 2′-carboxy ethylene disulfide (FIG. 14). Sincethe disulfide bonds are cleaved by glutathione inside the cell, it willstable in vascular system. In another embodiment, ketal groups arecleaved by the pH in endosomes in the cell. Since ketal groups arestable at physiological pH and above, this embodiment of the HBSPCM willbe stable in blood as well as during the chemical modification.

TABLE 1 CHARACTERISTICS OF JNK-1 AND JNK-2 HBSPCM POLYMERS Molecular No.cationic Average Zeta Kb C50 Blood weight units (R) of no. of potential(M−1) (nM) Rh compatibility Sample (Da) 4 amines each charges (mV) [1][1] (nm) at 1 mg/ml JNK-1 116 700  69 276 11.4 ± 2.9 1 108 10 10excellent JNK-2 48 000 28 112 11.1 ± 2.1 ND ND 4 excellent Protamine ~4500 — 21 ND ND ND ND poor [3] Tetramine   146 — 4 ND 3.6 105 2788 NDpoor (spermine) PEG-based 1.8 106 — ND ND 1 104 100000 ND ND linearcationic polymer [4] PEG = polyethylene glycols. Rh = hydrodynamicradius. ND = no data. [1] Binding affinity of polymers for ctDNA (ahighly anionic bio-macromolecule) in buffered water (pH 7.2) in thepresence of 50 mM NaCl at 25° C. [3] Showed complement activation,platelet activation, behaved like an anticoagulant, showed massivehemolysis. [4] Nisha et al. 2004 Langmuir. 20(6): 2386-2396.

In some embodiments, the binding affinity and charge density of HBSPCMscan be increased by incorporating hexa amine cationic units with sixnitrogens per unit instead of tetramine cationic units (FIG. 1). In someembodiments deca amine cationic units with 10 nitrogens per unit insteadof tetramine units (FIG. 1) are provided. In some embodiments, theHBSPCM structure is optimized using tosylation of HPG hydroxyl groupsfor the neutralization of heparin derivatives with a smaller number ofnegative charges than LMWH or UFH. In some embodiments, the HBSPCMstructure is optimized using reductive amination of HPG for theneutralization of heparin derivatives.

In some embodiments, the number of cationic units (R) is lower andvaries, each having four cationic amine groups per cationic unit andgiving a total charge per polymer of 4×R. For example, the number ofcationic units (R) can range from 4 to 24, giving a range of 16 to 96for charges per polymer. Table 2 lists the characteristics of suchpolymers, each with a molecular weight of 23 kDa, that have beensynthesized in some embodiments with different numbers of R groups, andhence charge per polymer, as well as different amine contents. In somesituations, JNK-8 can be considered a control because it is a HBSPCMpolymer without methylation of the amines, whereas in the other HBSPCMpolymers in Table 2 the primary amine groups are dimethylated.

TABLE 2 CHARACTERISTICS OF HBSPCMS AT 23,000 G/MOL WITH DIFFERENTCHARGES No. cationic units (R) Average no. of Average amine of 4 amineseach charges per polymer content (% N) JNK-4 4 16 3.0 JNK-5 5 20 5.0JNK-6 16 64 13 JNK-3 20 80 17 JNK-7 24 96 21 JNK-8 23 92 17.5 (Control)

In some embodiments, the molecular weight of the HBSPCMs is varied inaddition to the number of charges per polymer. For example, Table 3lists three additional polymers (JNK-9, JNK-10 and JNK-11) withmolecular weights of 9, 10, and 16 kDa, respectively, and number ofcharges per polymer of 32, 44 and 64, respectively.

TABLE 3 CHARACTERISTICS OF HBSPCMS WITH DIFFERENT MOLECULAR WEIGHTSMolecular Average No. Average no. Average weight cationic units (R) ofcharges amine Sample (Da) of 4 amines each per polymer content (% N)JNK-9  9 000 8 32 16.4 JNK-10 10 000 11 44 20.9 JNK-11 16 000 16 64 19.6

Cationic Groups

In some embodiments, the heparin binding polymer further includes acationic moiety. In some embodiments, the cationic moiety includes anamine group. In some embodiments, the cationic moiety can be arginine,secondary amine and primary amine groups, and/or N-methylated lysine. Insome embodiments, the amine is covalently attached to the polyol. Insome embodiments, the cationic moiety includes an arginine. In someembodiments, the amine group is a tetra-amine group, hexa-amine group,and/or a deca-amine group.

In some embodiments, the heparin binding polymer includes 1-300 cationicmoieties, for example 2-200, 3-150, 4-100, 5-50, 6-40, or 7-30. In someembodiments, the heparin binding polymer has 1 to 100 cationic moieties,for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 60,70, 80, 90, or 99 charges, including any amount between any of the twopreceding values. In some embodiments, the polymer has 4 to 23 cationicmoieties. In some embodiments, the polymer has 16 to 92 cationiccharges.

Cleavage Sites

In some embodiments the heparin binding polymer can include one or morecleavage site within the polymer. In some embodiments, there are 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 80, 100, or 200,including any range greater than any of the preceding values and anyrange defined between any two of the preceding values. In someembodiments, there are 1-10 cleavage sites. In some embodiments, theheparin binding polymer includes a second dendritic polyol, where the atleast one cleavage site links the first dendritic polyol to the seconddendritic polyol.

In some embodiments, the cleavage site can be a disulfide bond. In someembodiments the heparin binding polymer includes at least two sulfursthat form a disulfide bond. In some embodiments, the two sulfurs arepart of the structure of Formula (I):

where L₁ can be a first dendritic polyol, L₂ can be a second dendriticpolyol, L₃ can be a third dendritic polyol, L₄ can be a fourth dendriticpolyol, and L₅ can be a fifth dendritic polyol.

In some embodiments, the heparin binding polymer can include at leastone ketal group. In some embodiments there are 1, 2, 3, 4, 5, 6 7, 8, 9,10, 12, 15, 20, 25, 30, 50, 80, 100 or more ketal groups and/ordisulfide bonds in the heparin binding polymer. In some embodiments, theketal group is part of the structure of Formula (II):

L₁ can be a first dendritic polyol. L₂ can be a second dendritic polyol.L₃ can be a third dendritic polyol. L₄ can be a fourth dendritic polyol.

In some embodiments, the hyperbranched polyether polyol can furtherinclude one or more ketal groups. In some embodiments, the hyperbranchedpolyether polyol containing one or more ketal groups is degraded to formsmaller polymers of lower molecular weight. In some embodiments, thecleavage group can be that shown in FIG. 13. In some embodiments, thecleavage group can be that shown in FIG. 14.

In some embodiments, the hyperbranched polyether polyol can include oneor more disulphide bonds. In some aspects, the hyperbranched polyetherpolyol containing one or more disulphide bonds is degraded to formsmaller polymers of lower molecular weight.

Compositions and Pharmaceutical Formulations of Heparin Binding Polymers

In some embodiments, compositions including at least one HPG or heparinbinding polymer can be administered at a pharmacologically effectiveamount in a pharmaceutically acceptable excipient to a subjectpreviously administered with heparin, unfractionated heparin and/or aheparin derivative. Examples of heparin derivatives include, but are notlimited to, unfractionated heparin, low molecular weight heparins, ultralow molecular weight heparins, fondaparinux, idraparinux, heparinoidsand the like.

In some embodiments, a composition including at least two HPG polymerscan be administered to a subject at a pharmacologically effective amountin a pharmaceutically acceptable excipient previously administered withheparin or a heparin derivative. The HPG polymers can have differentmolecular weights, different functional groups, different PEG groupsizes, different charge density and the like, as described herein andknown in the art. Additionally, other agents can be coadministered withat least one HPG polymer. Examples of such agents may include protamine,protamine derivatives, pharmaceutical excipients, and the like.

In some embodiments, a heparin binding composition is provided. Thecomposition can include a first heparin binding polymer and apharmaceutically acceptable carrier. In some embodiments, thecomposition can include a first heparin binding polymer and an anionictherapeutic agent (e.g. heparins, methotrexate, insulin) for thecontrolled drug delivery. The heparin binding polymer can be any of theheparin binding polymer embodiments disclosed herein. In someembodiments, the heparin binding polymer includes a first dendriticpolyol and one or more cationic moieties attached to the first dendriticpolyol. In some embodiments, the composition further includes a secondheparin binding polymer. The second heparin binding polymer can bedifferent from the first heparin binding polymer. In some embodiments,the composition can include a protamine, a protamine derivative, or acombination thereof.

In some embodiments, the first heparin binding polymer is present in aunit dose. In some embodiments, the unit dose of the first heparinbinding polymer is between 10 mg per kg of subject to be treated and 200mg per kg of subject to be treated. In some embodiments, the subjectweighs between 1 kg and 200 kg, for example, between 5 kg and 150 kg,between 10 kg and 100 kg, or between 20 kg and 90 kg. In someembodiments, the composition includes between 100 mg and 40 g of thefirst heparin binding polymer, for example, between 500 mg and 20 g,between 1 g and 15 g, between 2 g and 10 g, or between 2 g and 5 g ofthe first heparin binding polymer.

In some embodiments, the heparin binding polymer can be prepared in amixture with a pharmaceutically acceptable carrier and/or excipient.Therapeutic compositions can be administered intravenously,subcutaneously, topically or via catheters When administeredsystemically, therapeutic compositions can be sterile, pyrogen-free andin a parenterally acceptable solution having due regard for pH,isotonicity, and stability. These conditions are known to those skilledin the art. In some embodiments, dosage formulations of the compoundsare prepared for storage or administration by mixing the compound havingthe desired degree of purity with physiologically acceptable carriers,excipients, or stabilizers. Such materials are non-toxic to therecipients at the dosages and concentrations employed, and includebuffers such as TRIS HCl, phosphate, citrate, acetate and other organicacid salts; antioxidants such as ascorbic acid; low molecular weight(less than about ten residues) peptides such as polyarginine, proteins,such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymerssuch as polyvinylpyrrolidinone; amino acids such as glycine, glutamicacid, aspartic acid, or arginine; monosaccharides, disaccharides, andother carbohydrates including cellulose or its derivatives, glucose,mannose, or dextrins; chelating agents such as EDTA; sugar alcohols suchas mannitol or sorbitol; counterions such as sodium and/or nonionicsurfactants such as TWEEN, PLURONICS or polyethyleneglycol.

Sterile compositions for injection can be formulated according toconventional pharmaceutical practice as described in Remington'sPharmaceutical Sciences (18^(th) ed, Mack Publishing Company, Easton,Pa., 1990). For example, dissolution or suspension of the activecompound in a vehicle such as water or naturally occurring vegetable oillike sesame, peanut, or cottonseed oil or a synthetic fatty vehicle likeethyl oleate or the like can be employed. Buffers, preservatives,antioxidants and the like can be incorporated according to acceptedpharmaceutical practice.

In some embodiments, a heparin composition is provided. The heparincomposition can include heparin and a heparin binding polymer. Theheparin binding polymer can include a first dendritic polyol and one ormore cationic moieties attached to the first dendritic polyol. In someembodiments, the heparin composition further includes blood or plasma.In some embodiments, the blood is human blood or the plasma is humanplasma. In some embodiments, the heparin is selected from the group oflow molecular weight heparin, fondaparinux, idraparinux, heparinoid, andany combination thereof.

In some embodiments, a method of making a heparin binding polymer isprovided. The method can include polymerizing a glycidol and amethylated PEG-epoxide, to form a hyperbranched polyglycerol, tosylatingthe hyperbranched polyglycerol to form a tosylated hyperbranchedpolyglycerol, and coupling the tosylated hyperbranched polyglycerol withat least one amine group, thereby making a heparin binding polymer.

In some embodiments, the amine group is coupled to the polymer through aprimary amino group. In some embodiments, following the coupling, theamino groups are methylated. In some embodiments, the amine group is atetra-amine group, a hexa-amine group, or a deca-amine group. In someembodiments, the tosylating process occurs in the presence of pyridineand at room temperature. In some embodiments, it occurs between 20 and30° C. In some embodiments, the polymerization process occurs at about95 degrees C. In some embodiments, it occurs between about 95° C. to120° C. and employs potassium methoxide. In some embodiments, it canemploy potassium tert-butoxide, sodium hydride, or sodium naphthalideinstead of potassium tert-butoxide, for example.

In some embodiments, molecules having cleavable bonds (HBSPCM polymerwith biodegradable disulfide linkages) from an initiator with disulfidelinkages. This disulfide containing initiator can be prepared, in someembodiments, by the reaction of the octamine with 2-hydroxy, 2′-carboxyethylene disulfide. HBSPCM polymer with biodegradable ketal linkages canbe prepared, in some embodiments, by using an initiator containing ketalgroups. This initiator can be prepared, in some embodiments by firstreacting ethylene diamine with 4 equivalents of glycidol, followed byreaction with 4-hydroxy-2-butanone.

Macromolecule

The term “macromolecule” as used herein denotes the presence of aprotective substance associated with the polyol core and/or cationicgroup. The term heparin binding polymer encompasses variousmacromolecules and is a broader genus to the possible types ofmacromolecules that can employ heparin binding polymers. Examples ofmacromolecules are shown in FIG. 1A and FIG. 3.

In some embodiments, the macromolecule includes a protective barrier(although it need not actually surround or completely surround the coreand/or cationic groups in all embodiments.

In some embodiments, the heparin binding macromolecule includes ahyperbranched polyglycidol core, at least one polyvalent cation attachedto the hyperbranched polyglycidol core, and at least one protectivemoiety attached to the hyperbranched polyglycidol core. In someembodiments, this protective moiety includes polyethylene glycol.

In some embodiments, the macromolecule has a polyethylene glycol contentbetween 50 and 95% by weight, e.g., 60-80%. In some embodiments themacromolecule has a polyethylene glycol content between 55% and 90%, 60%and 85%, 65% and 80%, 70% or 80% by weight. In some embodiments themacromolecule has a polyethylene glycol content of about 75% by weight.

In some embodiments, the polyvalent cation includes an amine group (suchas one or more of those shown in FIG. 1A). In some embodiments, theamine group includes a methylated amine. In some embodiments, the aminecan include an alkylated amine. In some embodiments, the amine groupincludes a methylated tetra-amine group, a methylated hexa-amine group,or a methylated deca-amine group. In some embodiments, the macromoleculehas a hydrodynamic radius of about 4 nm and about 10 nm.

Any of the other embodiments disclosed herein (such as the presence ofparticular cationic groups, cleavage sites, etc.), can be used incombination with any of the macromolecule embodiments.

Devices, Kits, Compositions, and Ex Vivo Techniques

In some embodiments, a heparin binding device is provided. The devicecan include a support and a heparin binding polymer immobilized on thesupport. In some embodiments, the heparin binding polymer is any of theheparin binding polymers and/or macromolecules described herein. In someembodiments, the heparin binding polymer includes a first dendriticpolyol and one or more cationic moieties attached to the first dendriticpolyol.

In some embodiments, the support includes a surface of one or morebeads. In some embodiments, the support includes metal, plastic, glass,or ceramic. In some embodiments, the support is a material that isbiocompatible. In some embodiments, the material can be polyurethane,PVC, silicone, etc.

In some embodiments, the device also includes a fluid flow pathconfigured to allow a fluid to flow through the fluid flow path whilecoming in contact with the heparin binding polymer on the support.

In some embodiments, the device also includes a pump configured to pumpblood through the fluid flow path. In some embodiments, the pump is aperistaltic pump.

In some embodiments, the support upon or in which the heparin bindingpolymer is associated is, or is part of, a removable cartridge. In someembodiments, the removable cartridge allows for the replacement of oneset of heparin binding polymers with a different set. In someembodiments, the cartridge allows for the ready replacement of a usedset of heparin binding polymers with a new set of polymers.

In some embodiments, the support is part of the removable cartridge. Insome embodiments, the support in the cartridge is configured so as toallow the support to be exposed to a fluid in the fluid flow path. Insome embodiments, the heparin binding polymer is attached to a surfaceof the cartridge. In some embodiments, the removable cartridge, whenengaged with the device, forms part of the flow path or an outer surfaceof the flow path. In some embodiments, the cartridge is configured suchthat the fluid flows over the support section of the cartridge. In someembodiments, the cartridge is configured such that the fluid flowsthrough the support section of the cartridge (e.g., the support can be afilter or screen). In some embodiments, the cartridge is configured suchthat the fluid flows around and/or to the side of the support section ofthe cartridge (e.g., the support can be a structure that is insertedinto the flow path itself).

In some embodiments, the device will include blood when in use. In someembodiments, the blood is mammalian, such as human, pig, horse, cow,mouse, etc.

In some embodiments, the fluid flow path includes a first inlet, throughwhich unprocessed blood can enter the flow path, and an outlet, throughwhich processed blood can exit the heparin binding device.

In some embodiments, the device includes a reservoir in fluidcommunication with a second inlet. In some embodiments, the reservoirincludes one or more plasma constituents. In some embodiments, thereservoir includes one or more blood constituents. In some embodiments,the plasma constituents or the blood constituents include a non-heparinmolecule that binds to the heparin binding polymer. In some embodiments,the reservoir contains a medicine for the treatment of at least one ofthe following: acute coronary syndrome, atrial fibrillation, cardiacbypass, extracorporeal membrane oxygenation, pancreatitis, wounds,hemofiltration, inflammation, cancer and burns.

In some embodiments, the device includes tubing, or is the tubingitself, and the inside of the tubing is coated inside with a heparinbinding polymer.

In some embodiments, HPG polymers can be used in an extracorporealcircuit having heparin or heparin derivatives induced into the blood forreuse in a subject, where the extracorporeal device contains HPGpolymers to neutralize the heparin or heparin derivatives in the bloodbefore returning it to the subject. In some embodiments, the HPGpolymers are HBSPCM polymers attached to the part of the extracorporealdevice coming into contact with blood.

In some embodiments, a kit is provided. The kit can include a firstcontainer that includes heparin and a second container that includes aheparin binding polymer. In some embodiments, the heparin bindingpolymer includes a first dendritic polyol and one or more cationicmoieties attached to the first dendritic polyol. In some embodiments,the amount of heparin binding polymer included is sufficient to bind tothe amount of heparin included. In some embodiments, the heparin bindingpolymer is divided into unit dosage amounts, such that the amount ofheparin binding polymer in any single dose amount is sufficient to bindto a standard dose of heparin. In some embodiments, the kit can includea needle and/or tubing for withdrawing blood from a subject. In someembodiments, the kit can include cartridges and/or a device as describedherein. In some embodiments, the kit can include one or more syringes.In some embodiments, the syringe is sized so as to be capable ofaccepting a unit dose of heparin and/or the heparin binding polymer. Insome embodiments, a unit dose of the heparin binding polymer isprefilled into the syringe. In some embodiments, the kit includescomponents for intravenous administration of the polymer. In someembodiments, material for measuring thrombin and/or fXa activity canalso be included.

In some embodiments, a controlled release delivery device is provided.The device can include a surface and a heparin binding polymer attachedto the surface. The heparin binging polymer can include a firstdendritic polyol and one or more cationic moieties attached to the firstdendritic polyol. In some embodiments, any of the polymers describedherein can be employed. In some embodiments, the controlled releasedelivery device can also include heparin. In some embodiments, theheparin is bound to the heparin binding polymer. In some embodiments,the type of heparin can be any of the types described herein. In someembodiments, the amount of heparin is adequate for a specifictherapeutic use. In some embodiments, the delivery device is adequatelysterile for the use of the device in a subject, such as a human ormammal. In some embodiments, the controlled release delivery deviceallows for an extended release of heparin in a subject. In someembodiments, the controlled release delivery device allows for a loweramount of heparin to be administered to a subject. In some embodiments,the controlled release delivery device allows for relatively localizeddelivery of heparin. In some embodiments, instead of heparin, an anionicdrug, such as methotrexate, phenoxymethyl penicillin, insulin,indomethacin, diclofenac, etc. can be used, thereby providing acontrolled release anionic delivery device.

In some embodiments, a controlled release heparin composition isprovided. In some embodiments, the composition can include a heparinbinding polymer. In some embodiments, the heparin binding polymer caninclude a first dendritic polyol and one or more cationic moietiesattached to the first dendritic polyol. In some embodiments, thecontrolled release heparin composition can also include heparin. In someembodiments, the heparin is bound to the heparin binding polymer. Insome embodiments, the type of heparin can be any of the types describedherein. In some embodiments, the amount of heparin is adequate for aspecific therapeutic use. In some embodiments, the composition isadequately sterile for its application to a subject, such as a human ormammal. In some embodiments, the controlled release heparin bindingcomposition allows for an extended release of heparin in a subject. Insome embodiments, the controlled release heparin binding compositionallows for a lower amount of heparin to be administered to a subject. Insome embodiments, the controlled release heparin binding compositionallows for relatively localized delivery of heparin. In someembodiments, instead of heparin, an anionic drug, such as methotrexate,phenoxymethyl penicillin, insulin, indomethacin, diclofenac, etc. can beused, thereby providing a controlled release anionic drug composition.

Methods of Using Heparin Binding Polymers

In some embodiments, methods are provided for neutralizing heparin,heparin derivatives, and/or heparinoids. The method can includeadministering a composition having at least one species of heparinbinding polymer and/or hyperbranched polyether polyol polymers to asubject. The subject can have had a previous administration of heparinor heparin derivative.

In some embodiments, there is provided a method for neutralizing heparinor heparin derivatives, the method including directing the blood from asubject through an extracorporeal circuit that contains at least onespecies of heparin binding polymer and/or hyperbranched polyether polyolpolymer before returning the blood to the subject for reuse. The subjectmay have had a previous administration of heparin, heparin derivativeand/or heparinoids.

In some embodiments, a method of counteracting heparin in a subject isprovided. In some embodiments, the method includes administering aheparin binding polymer to a subject. In some embodiments, the heparinbinding polymer includes a first dendritic polyol and one or morecationic moieties attached to the first dendritic polyol. In someembodiments, the heparin binding protein binds to heparin and therebycounteracts heparin in the subject. In some embodiments, the heparinbinding polymer further includes polyethylene glycol covalently attachedto the heparin binding polymer. In some embodiments, the cationic moietyis an amine group. In some embodiments, the amine group is a tetra-aminegroup, a hexa-amine group, or a deca-amine group. In some embodiments,the heparin binding polymer is any one or more of the heparin bindingpolymers and/or macromolecules provided herein.

In some embodiments, the method can include identifying a subject whohas received an excess amount of heparin, wherein the identifying occursbefore the heparin binding polymer is administered to the subject. Insome embodiments, this can be done by identifying a subject who hasreceived heparin in a clinical setting. In some embodiments, the excessamount of heparin was administered to the subject. In some embodiments,the excess amount of heparin was administered for the maintenance of anintravenous catheter or other hospital or medical care related treatmentor service. In some embodiments, the excess amount of heparin wasadministered to treat one or more of the following: acute coronarysyndrome, atrial fibrillation, cardiac bypass, extracorporeal membraneoxygenation, pancreatitis, hemofiltration, cancer therapy, or burns.

In some embodiments, the method includes identifying a subject thatwould benefit from reducing a level of exogenous heparin that is presentin the subject's blood. In some embodiments, the subject will benefitfrom an accelerated removal of exogenous heparin from the subject.

In some embodiments, the method is performed in vivo. In someembodiments, the method is performed ex vivo. In some embodiments, bloodis taken from a subject and heparin is removed from the blood and/orplasma via the heparin binding polymer. In some embodiments, the subjectis a human. In some embodiments, the subject is not a human. In someembodiments, the subject is a dog, a cat, a horse, a cow, a pig, amouse, a rat, a rabbit, a monkey, or a guinea pig.

In some embodiments, the heparin binding polymer can be administeredsubcutaneously. In some embodiments, the heparin binding polymer can beadministered intravenously.

In some embodiments, a method of processing a subject's blood isprovided. The method can include providing a heparin binding device. Theheparin binding device can be one described herein. In some embodiments,the device can include a support and a heparin binding polymerimmobilized on the support. The heparin binding polymer can include afirst dendritic polyol and one or more cationic moieties attached to thefirst dendritic polyol. The method can further include withdrawing bloodfrom a subject, contacting the blood to the heparin binding polymer, andreturning at least part of the blood to the subject, thereby processingthe subject's blood. In some embodiments, the process is done withand/or to the subject's plasma instead of blood. In some embodiments,the method further includes isolating plasma from the subject, treatingthe plasma to remove heparin, and then returning the plasma to the samesubject and/or a different subject. In some embodiments, the subject'sblood contains heparin. In some embodiments, the heparin is exogenousheparin. In some embodiments, the exogenous heparin binds to the heparinbinding polymer and is thereby at least partially removed from thesubject's blood.

In some embodiments, the method further includes the step of selecting aheparin binding polymer based upon the type of exogenous heparinadministered to the subject. In some embodiments, the heparin bindingpolymer of a particular molecular weight is selected based upon the typeof exogenous heparin. In some embodiments, this is done based upon thebinding strength and neutralization capacity of the exogenous heparin.In some embodiments, a heparin binding polymer having a particularnumber of cationic groups is selected based upon the type of exogenousheparin. In some embodiments, the heparin binding polymer works for amajority of heparins or all heparins. In some embodiments, the heparinbinding polymer works for negatively charged heparins, includingderivatives thereof.

In some embodiments, returning at least part of the blood to the subjectfurther includes fortifying the blood with one or more bloodconstituents that also bind to the heparin binding polymer. In someembodiments, the fortifying is performed after the blood has contactedthe heparin binding polymer. In some embodiments, the blood can befortified with negatively charged coagulation factors and/or proteins.

In some embodiments, a method of concentrating heparin is provided. Insome embodiments, the method includes providing a support and a heparinbinding polymer immobilized on the support. The heparin binding polymercan be any of the heparin binding polymers disclosed herein. In someembodiments, the heparin binding polymer includes a first dendriticpolyol and one or more cationic moieties attached to the first dendriticpolyol. In some embodiments, the method also includes contacting a firstfluid that includes heparin with the heparin binding polymer and flowingthe fluid off of the heparin binding polymer. This can concentrate theheparin.

In some embodiments, the first fluid includes blood. In someembodiments, the blood is human. In some embodiments, the fluid includesa cell lysate or other fluid from the manufacturing process ofmanufacturing heparin.

In some embodiments, the method further includes the process of removingthe heparin from the heparin binding polymer. In some embodiments, theheparin is removed by flowing a second fluid including an ionic solutionover the heparin binding polymer. In some embodiments, the first supportis part of an affinity chromatography device. In some embodiments, thefirst fluid is derived from mucosal tissue from a meat animal. In someembodiments, the mucosal tissue from a meat animal is either porcineintestine, bovine lung, from cell culture, or a combination thereof.

In some embodiments, flowing the fluid off of the heparin bindingpolymer removes at least one impurity, thereby purifying the heparin.

In some embodiments, heparin binding polymer is used for the controlleddelivery of anionic therapeutic agents such as heparins, methetrixate,insulin or similar for the treatments of VTE, cancer, inflammation ordiabetes.

In some embodiments, the HBSPCM polymers are stable from −80° C. to 120°C. In some embodiments, heparin solutions are stable in the temperaturerange 4° C. to body temperature. In some embodiments, the methods canoccur in part or in whole within one or both of the above temperatureranges.

In some embodiments, a polymer pad is provided. The polymer pad can becoated with any of the polymers disclosed herein. In some embodiments,this can be used as a controlled delivery platform for heparin baseddrugs or anionic drugs and/or for the local delivery of heparin. In someembodiments, the heparin binding polymer can be employed as apreventative, and thus can be applied prophylactically. In someembodiments the heparin binding polymers can be employed to treatedheparin sulphate related disorders. For example, as a treatment orprophylactic for subjects who overexpress heparin sulphate.

EXAMPLES Example 1 Synthesis of HPG-PEG-23K Precursor Polymer

The present example outlines a method of producing 23 kDa HBSPCMpolymers; the synthesis steps include the following. Firstly, aHPG-PEG-23K precursor polymer was synthesized using the following steps.A three-necked round bottomed flask was cooled under vacuum and filledwith argon. To this, 1,1,1-Tris(hydroxymethyl)propane (TMP, 0.480 g) andpotassium methylate (25 wt % solution in methanol, 0.440 mL) were addedand stirred for 30 minutes. Methanol was removed under vacuum for 4hours. The flask was heated to 95° C. and glycidol (10 mL) was addedover a period of 15 hours. After complete addition of monomer, thereaction mixture was stirred for additional 3 hours. MPEG-epoxide-400(32 mL) was added over a period of 12 hours. The reaction mixture wasstirred for additional 4 hours. The polymer was dissolved in methanol,passed through Amberlite IR-120H resin to remove the potassium ions andtwice precipitated from diethyl ether. The polymer was dissolved inwater and dialyzed against water using MWCO-1000 membrane for 3 dayswith periodic changes in water.

Example 2 Synthesis of JNK-5 Polymer

The JNK-5 polymer was synthesized from the HPG-PEG-23K precursor polymerdescribed in the previous paragraph as follows. HPG-PEG-23K (0.5 g) wasdissolved in 10 mL of pyridine. To this, p-toluene sulfonyl chloride(0.5 g) was added and stirred at room temperature for 24 hours. Pyridinewas removed by rotary evaporation; the polymer was dissolved in 0.1 NHCl and dialyzed overnight. The polymer was isolated by freeze drying.HPG-PEG-Tos-23K-1 (0.4 g; polymer from previous step) andtris(2-aminoethylamine) (2 mL) were dissolved in 1,4-dioxane (10 mL) andrefluxed for 24 hours. Dioxane was removed under vacuum, the polymer wasdissolved in minimum amount of methanol and precipitated twice fromdiethyl ether. Polymer was dissolved in water and dialyzed against waterusing MWCO-1000 membrane for 2 days. The resulting polymer solution wasadded to a mixture of formaldehyde (3 mL) and formic acid (3 mL) at 0°C. The reaction mixture was refluxed overnight. After cooling to roomtemperature, the pH of the solution was adjusted to 10 using NaOH andthe polymer was extracted with dichloromethane. Dichloromethane wasremoved under vacuum. The polymer was dissolved in water and dialyzedusing MWCO-1000 membrane for 3 days. The yield of JNK-5 polymers wasshown to be 0.3 g, and the amine content (by conductometric titration)was 4.8 mol %.

Example 3 Synthesis of JNK-3 Polymer

The HPG-PEG-23k precursor polymer (0.5 g) was dissolved in 10 mL ofpyridine. To this, p-toluene sulfonyl chloride (1 g) was added andstirred at room temperature for 24 hours. Pyridine was removed by rotaryevaporation; the polymer was dissolved in 0.1 N HCl and dialyzedovernight. The polymer was isolated by freeze drying. HPG-PEG-Tos-23K-2(0.5 g; polymer from previous step) and tris(2-aminoethylamine) (4 mL)were dissolved in 1,4-dioxane (10 mL) and refluxed for 24 hours. Dioxanewas removed under vacuum, the polymer was dissolved in minimum amount ofmethanol and precipitated twice from diethyl ether. Polymer wasdissolved in water and dialyzed against water using MWCO-1000 membranefor 2 days. The resulting polymer solution was added to a mixture offormaldehyde (3 mL) and formic acid (3 mL) at 0° C.

The reaction mixture was refluxed overnight. After cooling to roomtemperature, the pH of the solution was adjusted to 10 using NaOH andthe polymer was extracted with dichloromethane. Dichloromethane wasremoved under vacuum; the polymer dissolved in water and dialyzed usingMWCO-1000 membrane for 3 days. The yield of JNK-3 polymers was shown tobe 0.3 g, and the amine content (by conductometric titration) was 16.9mol %.

Example 4 Synthesis of JNK-4 Polymer

The HPG-PEG-23k precursor polymer (0.5 g) was dissolved in 10 mL ofpyridine. To this, p-toluene sulfonyl chloride (0.25 g) was added andstirred at room temperature for 24 hours. Pyridine was removed by rotaryevaporation; the polymer was dissolved in 0.1 N HCl and dialyzedovernight. The polymer was isolated by freeze drying. HPG-PEG-Tos-23K(0.4 g; polymer from previous step) and tris(2-aminoethylamine) (2 mL)were dissolved in 1,4-dioxane (10 mL) and refluxed for 24 hours. Dioxanewas removed under vacuum, the polymer was dissolved in minimum amount ofmethanol and precipitated twice from diethyl ether. Polymer wasdissolved in water and dialyzed against water using MWCO-1000 membranefor 2 days. The resulting polymer solution was added to a mixture offormaldehyde (2 mL) and formic acid (2 mL) at 0° C.

The reaction mixture was refluxed overnight. After cooling to roomtemperature, the pH of the solution was adjusted to 10 using NaOH andthe polymer was extracted with dichloromethane. Dichloromethane wasremoved under vacuum; the polymer dissolved in water and dialyzed usingMWCO-1000 membrane for 3 days. The yield of JNK-4 polymers was shown tobe 0.25 g, and the amine content (by conductometric titration) was 3 mol%.

Example 5 Synthesis of JNK-6 and JNK-7 Polymers

The HPG-PEG-23k precursor polymer was used as starting polymer for thesynthesis of the JNK-6 and JNK-7 polymers. To change the amine contentof the polymer, different amounts of p-toluene sulfonyl chloride wereused in the tosylation step. By controlling the amount of tosyl groupsincorporated, the amine coupling was changed. All other experimentalconditions for synthesis were the same as for the JNK-3 polymer.

Example 6 Synthesis of JNK-8 Polymer

The HPG-PEG-23k precursor polymer was used as starting polymer for thesynthesis of the JNK-8 polymers. We followed similar experimentalconditions and reagents as for the JNK-3 polymer except the lastdimethylation using formaldehyde and formic acid, which was notperformed. JNK-8 is a primary amine containing polymer without thedimethylation of amines.

Example 7 Synthesis of JNK-9 Polymer

Firstly, a precursor polymer HpG-PEG-9K was synthesized as follows. Athree-necked round bottomed flask was cooled under vacuum and filledwith argon. To this, 1,1,1-Tris(hydroxymethyl)propane (TMP, 0.240 g) andpotassium methylate (25 wt % solution in methanol, 0.220 mL) were addedand stirred for 30 minutes. Methanol was removed under vacuum for 4hours. The flask was heated to 95 C and glycidol (3 mL) was added over aperiod of 15 hours. After complete addition of monomer, the reactionmixture was stirred for additional 3 hours. MPEG-epoxide-400 (10 mL) wasadded over a period of 12 hours. The reaction mixture was stirred foradditional 4 hours. The polymer was dissolved in methanol, passedthrough Amberlite IR120H resin to remove the potassium ions and twiceprecipitated from diethyl ether. The polymer was fractional precipitatedin methanol/ether mixture (1:25 v/v) to obtain HPG-PEG-9k polymer. Thepolymer was dissolved in water and dialyzed against water usingMWCO-1000 membrane for 3 days with periodic changes in water.

Secondly, a precursor polymer HPG-PEG-Amine-9K was synthesized asfollows. HPG-PEG-9K (1.0 g) was dissolved in 20 mL of pyridine. To this,p-toluene sulfonyl chloride (5.0 g) was added and stirred at roomtemperature for 24 hours. Pyridine was removed by rotary evaporation;the polymer was dissolved in 0.1 N HCl and dialyzed overnight. Thepolymer was isolated by freeze drying. HPG-PEG-Tos-9K (0.65 g; polymerfrom previous step) and tris(2-aminoethylamine) (3 mL) were dissolved in1,4-dioxane (25 mL) and refluxed for 24 hours. Dioxane was removed undervacuum; the polymer was dissolved in minimum amount of methanol andprecipitated twice from diethyl ether. Polymer was dissolved in waterand dialyzed against water using MWCO-1000 membrane for 2 days.

The above polymer solution was added to a mixture of formaldehyde (3 mL)and formic acid (3 mL) at 0° C. The reaction mixture was refluxedovernight. After cooling to room temperature, the pH of the solution wasadjusted to 10 using NaOH and the polymer was extracted withdichloromethane. Dichloromethane was removed under vacuum; the polymerdissolved in water and dialyzed using MWCO-1000 membrane for 3 days. Theyield was 0.4 g and the amine content (by conductometric titration) was16.35 mol %.

Example 8 Synthesis of JNK-10 Polymer

Firstly, a precursor polymer HPG-PEG-10K was synthesized as follows. Athree-necked round bottomed flask was cooled under vacuum and filledwith argon. To this, 1,1,1-Tris(hydroxymethyl)propane (TMP, 0.240 g) andpotassium methylate (25 wt % solution in methanol, 0.220 mL) were addedand stirred for 30 minutes. Methanol was removed under vacuum for 4hours. The flask was heated to 95° C. and glycidol (2.5 mL) was addedover a period of 15 hours. After complete addition of monomer, thereaction mixture was stirred for additional 3 hours. MPEG-epoxide-400 (9mL) was added over a period of 12 hours. The reaction mixture wasstirred for additional 4 hours. The polymer was dissolved in methanol,passed through Amberlite IR-120H resin to remove the potassium ions andtwice precipitated from diethyl ether. The polymer was fractionalprecipitated in methanol/ether mixture (1:10 v/v) to obtain HPG-PEG-10kpolymer. The polymer was dissolved in water and dialyzed against waterusing MWCO-1000 membrane for 3 days with periodic changes in water.

Secondly, a precursor polymer HPG-PEG-Amine-10K was synthesized asfollows. HPG-PEG-10K (2.4 g) was dissolved in 25 mL of pyridine. Tothis, p-toluene sulfonyl chloride (8.0 g) was added and stirred at roomtemperature for 24 hours. Pyridine was removed by rotary evaporation;the polymer was dissolved in 0.1 N HCl and dialyzed overnight. Thepolymer was isolated by freeze drying. HPG-PEG-Tos-10K (2.8 g; polymerfrom the previous step) and tris(2-aminoethylamine) (8 mL) weredissolved in 1,4-dioxane (25 mL) and refluxed for 24 hours. Dioxane wasremoved under vacuum; the polymer was dissolved in minimum amount ofmethanol and precipitated twice from diethyl ether. Polymer wasdissolved in water and dialyzed against water using MWCO-1000 membranefor 2 days.

The above polymer solution was added to a mixture of formaldehyde (6 mL)and formic acid (6 mL) at 0° C. The reaction mixture was refluxedovernight. After cooling to room temperature, the pH of the solution wasadjusted to 10 using NaOH and the polymer was extracted withdichloromethane. Dichloromethane was removed under vacuum; the polymerdissolved in water and dialyzed using MWCO-1000 membrane for 3 days. Theyield was 1 g and the amine content (by conductometric titration) was20.9 mol %.

Example 9 Synthesis of JNK-11 Polymer

Firstly, a precursor polymer HPG-PEG-16K was synthesized as follows. Athree-necked round bottomed flask was cooled under vacuum and filledwith argon. To this, 1,1,1-Tris(hydroxymethyl)propane (TMP, 0.240 g) andpotassium methylate (25 wt % solution in methanol, 0.220 mL) were addedand stirred for 30 minutes. Methanol was removed under vacuum for 4hours. The flask was heated to 95 C and glycidol (3 mL) was added over aperiod of 15 hours. After complete addition of monomer, the reactionmixture was stirred for additional 3 hours. MPEG-epoxide-400 (10 mL) wasadded over a period of 12 hours. The reaction mixture was stirred foradditional 4 hours. The polymer was dissolved in methanol, passedthrough Amberlite IR-120H resin to remove the potassium ions and twiceprecipitated from diethyl ether. The polymer was fractional precipitatedin methanol/ether mixture (1:5 v/v) to obtain HPG-PEG-16k polymer. Thepolymer was dissolved in water and dialyzed against water usingMWCO-1000 membrane for 3 days with periodic changes in water.

Secondly, a precursor polymer HPG-PEG-Amine-16K was synthesized asfollows. HPG-PEG-16K (1.7 g) was dissolved in 20 mL of pyridine. Tothis, p-toluene sulfonyl chloride (6.0 g) was added and stirred at roomtemperature for 24 hours. Pyridine was removed by rotary evaporation;the polymer was dissolved in 0.1 N HCl and dialyzed overnight. Thepolymer was isolated by freeze drying. HPG-PEG-Tos-16K (2 g; polymerfrom the previous step) and tris(2-aminoethylamine) (6 mL) weredissolved in 1,4-dioxane (25 mL) and refluxed for 24 hours. Dioxane wasremoved under vacuum; the polymer was dissolved in minimum amount ofmethanol and precipitated twice from diethyl ether. Polymer wasdissolved in water and dialyzed against water using MWCO-1000 membranefor 2 days.

The above polymer solution was added to a mixture of formaldehyde (6 mL)and formic acid (6 mL) at 0° C. The reaction mixture was refluxedovernight. After cooling to room temperature, the pH of the solution wasadjusted to 10 using NaOH and the polymer was extracted withdichloromethane. Dichloromethane was removed under vacuum; the polymerdissolved in water and dialyzed using MWCO-1000 membrane for 3 days. Theyield was 1.3 g and the amine content (by conductometric titration) was19.62 mol %.

Example 10 Binding Studies with Heparin and Heparin Derivatives

To demonstrate the utility and performance of HBSPCMs for neutralizingheparin, binding studies were performed with both unfractionated heparin(UFH) and low molecular weight heparin (LMWH) in in vitro human bloodstudies and in rats. HBSPCMs were also characterized for their abilityto induce anticoagulation and clots in blood.

UFH and LMWHs are neutralized significantly more efficiently with theHBSPCMs than with protamine. Furthermore, HBSPCMs do not show anyadverse effects on blood coagulation, platelet activity, complementactivation, hemolysis and cytotoxicity while protamine and othercationic macromolecules do have adverse side effects.

Table 4 demonstrates a comparison of two embodiments of the polymer(JNK-1 and JNK-2) in terms of their ability to neutralize unfractionatedheparin and low molecular weight heparin (LMWH), and shows a comparisonwith equivalent values for protamine. Both JNK-1 and JNK-2 result ingreater neutralization of unfractionated heparin at two doses (50 and100 μg/ml), Tinzaparin (LMWH) and Enoxaparin (LMWH).

TABLE 4 NEUTRALIZATION EFFECT OF JNK-1 AND JNK-2 POLYMERS Anti Xa AntiXa Anti Xa Anti Xa Neutralization (%) Neutralization (%) Neutralization(%) Neutralization (%) of UFH at 100 of Tinzaparin at 100 of Enoxaparinat 100 of UFH at 50 Sample μg/ml [1] μg/ml [1] μg/ml [1] μg/ml [1] JNK-189 ± 3 90 ± 2 81 ± 3 86 JNK-2 90 ± 2 92 ± 4 89 ± 3 90 Protamine 85 ± 077 ± 1 73 ± 4 84 UFH = unfractionated heparin. [1] One IU UFH or LMWH(Enoxaparin or Tinzaparin) per ml in platelet poor plasma.

Example 11 JNK-1 Neutralization of UFH

In one example, a heparin binding synthetic polyvalent cationicmacromolecule JNK-1 can neutralize UFH in human blood under in vitroconditions (FIGS. 4A and 4B). Citrate plasma was anti-coagulated with1.0 U/ml UFH (UFH control) and was titrated with differentconcentrations of JNK-1 polymers (0.009 to 8.69 nanomoles/ml) andprotamine (0.2 to 222.2 nanomoles/ml). The activated partialthromboplastin time (APTT) was measured for each sample and is shown inFIGS. 4A and 4B. These results show that JNK-1 polymers effectivelyneutralized UFH (FIG. 4A) and do not cause anticoagulant effect even athigh concentration, unlike protamine (FIG. 4B). JNK-1 polymers showedgreater UFH neutralization capacity as compared to protamine.

Example 12 JNK-1 Neutralization of LMWH Tinzaparin

The present example demonstrates that JNK-1 polymers can neutralize LMWHTinzaparin in human blood in vitro (FIGS. 4A and 4B). Citrate plasma wasanti-coagulated with 2.0 U/ml LMWH (LMWH control) and was titrated withdifferent concentrations of JNK-1 polymers (0.009 to 8.69 nanomoles/ml)(FIG. 4A) and protamine (0.2 to 222.2 nanomoles/ml) (FIG. 4B). Theactivated partial thromboplastin time (APTT) was measured for eachsample, as shown in FIGS. 4A and 4B. Results show that JNK-1 effectivelyneutralizes Tinzaparin and does not cause any anticoagulant effect ateven higher concentration, unlike protamine. Unlike protamine, JNK-1polymers completely neutralized the anticoagulation of LMWH and showedgreater efficiency.

Example 13 Percentage of UFH and LMWH Tinzaparin Neutralization

The present example demonstrates the percentage of UFH and LMWHTinzaparin neutralization calculated from an activated partialthromboplastin time (APTT) assay by JNK-1 or protamine. FIG. 5A showsthe JNK-1 polymer neutralization of UFH. FIG. 5B shows the protamineneutralization of UFH. FIG. 5C shows the JNK-1 polymer neutralization ofLMWH. FIG. 5D shows the protamine neutralization of LMWH. The IC₅₀ ofUFH neutralization by JNK-1 polymers is 0.1 nanomoles and of Tinzaparinis 0.2 nanomoles, which is a few orders lower (more efficient) thanprotamine. JNK-1 completely neutralizes UFH and Tinzaparin.

Example 14 Thromboelastography in the Presence of Various HeparinBinders

This example examines thromboelastography in the presence of variousheparin binders. Thromboelastography (TEG) provides an in vitro methodfor monitoring of the evolution of the viscoelastic properties andcoagulation kinetics of blood as clotting proceeds. The principlecomponents of the TEG are a cylindrical cup and a bob suspended in thecup from a weak torsion wire. The warmed cup oscillates over a 10 secondperiod through an angle of 4° 45′. Blood is added and a torque istransmitted to the bob as the gel forms. The rate of clot formation, thestrength of the clot, and the rate of clot lysis are reflected in theshape traced out by the maxima in the bob oscillation as a function oftime (the trace of the TEG). The clot's physical properties, i.e. rateof clot formation, clot strength, and stability are dependent on theinteraction of fibrinogen, platelets, and plasma proteins. TEG studiesyield the cumulative effect of several components of coagulation (globalhomeostasis) as a function of time.

To carry out TEG experiments, citrate anti-coagulated whole blood withheparin or heparin derivatives added are incubated with buffered HBSPCMpolymers (sodium citrate or saline) for 1 minute before recalcificationat 37° C. Clot formation kinetics are monitored in a TEG machine (350 μlwhole blood+35 μl polymer or control solution) at 37° C.

JNK-1 polymers can neutralize UFH and LMWH Tinzaparin, as demonstratedby using a TEG measurement of human whole blood (FIG. 6). TEG traces ofdifferent experiments are given. JNK-1 polymer neutralization of UFH isshown in FIG. 6A. Protamine neutralization of UFH is shown in FIG. 6B.JNK-1 polymer neutralization of Tinzaparin is shown in FIG. 6C.Protamine neutralization of Tinzaparin is shown in FIG. 6D. The resultsdemonstrate the ability of JNK-1 to neutralize UFH and Tinzaparin evenat higher concentration, unlike protamine. There was no clot formationobserved in presence of either UFH or Tinzaparin alone.

Example 15 JNK-1 Polymers can Neutralize UFH and LMWH Tinzaparin

The present example demonstrates that JNK-1 polymers can neutralize UFHand LMWH Tinzaparin, as demonstrated by using a TEG measurement of humanwhole blood (FIGS. 7A and 7B). The coagulation time (FIG. 7A) andmaximum clot strength, or maximum amplitude (MA) (FIG. 7B) are shown.Citrated whole blood was added to UFH or Tinzaparin (0.5 U/ml) andre-calcified at 37° C. in a TEG cup and monitored using a TEG machine.JNK-1 polymers or protamine at different concentrations were used asneutralizing agents.

The results demonstrate that JNK-1 polymers effectively neutralizeanticoagulation effect of both UFH and Tinzaparin. Protamine at a higherconcentration (0.25 mg/ml) did not form a stable clot (see maximumamplitude (MA) data) with Tinzaparin. There was no clot formationobserved in presence of either UFH or Tinzaparin alone.

Example 16 Electrostatic Charge Neutralization of UFH and LMWHTinzaparin by JNK-1 Polymers

This example demonstrates the electrostatic charge neutralization of UFHand LMWH Tinzaparin by JNK-1 polymers (results shown in FIG. 9). Theelectrophoretic mobilities of heparin/JNK-1 complexes formed by mixingthe different ratios of heparin and JNK-1 polymers were measured. JNK-1polymers neutralize UFH and Tinzaparin at similar concentrations. Theresults are presented in FIG. 9.

Example 17 Complement Activation

The present example examines complement activation due to heparinbinding polymers. Complement activation is one of the major adverse sideeffects associated with the clinical use of protamine in the reversal ofanticoagulation in cardiopulmonary bypass and other surgical conditions(Shastri et al. 1997 Cardiovasc Surg. 114(3):482-8). Complementactivation Induced by various biomaterials has been studied (Lamba etal. 1999. Biomaterials 21:89; Kidane et al. 1999. J Biomed Mate Res48:640; Lim et al. 1993. Biomaterials 14:537; Payne et al. 1987. JBiomed Mater Res 21:843; Jantova et al. 1991. Complement andInflammation 8:61). Examples of polymers which cause Complementactivation include dextran, regenerated cellulose, sephadex, nylon,poly(methylmethacrylate), poly(propylene), poly(acrylamide),poly(hydroxyethylmethacrylate), plasticised PVC. Complement activationis not a property of all polymers however. For example,poly(N-vinylpyrrolidone) does not cause complement activation (Beillatet al. 1984. ASAIO J 7:57).

To assess complement activation, the activation of the complementcomponent C3 was monitored by measuring the formation of its activationpeptides using a commercial C3a enzyme immunoassay kit (Quidel, SanDiego, Calif.), following manufacturer's instructions. The C3aconcentrations (in ng/ml) were calculated using a standard curve withnet absorbance values plotted on the y-axis for each C3a concentrationindicated on the x-axis.

Complement activation upon interaction of JNK-1 polymers with plateletpoor plasma (10 mg/ml and 1 mg/ml) is shown in FIG. 10A at 37° C. for 1hour at 9:1 dilution of platelet poor plasma. Plasma incubated withsaline as a control and insulin (a potent complement activator) aspositive control are also given. Results show that JNK-1 polymers do notinitiate complement activation.

FIG. 20 demonstrates the complement activation by the HBSPCM polymersdescribed in Table 2 (JNK-3, JNK-4, JNK-5, JNK-6, JNK-7 andJNK-8-control) in comparison to protamine and a saline control.Complement activation is shown upon interaction of the HBSPCM polymersin platelet poor plasma (PPP) at 37° C. for 1 hour at a 9:1 dilution ofPPP. At 0.5 mg/mL, the HBSPCM polymers all had reduced complementactivation as compared to protamine. At 0.05 mg/mL, all but one HBSPCMpolymer (JNK-7) had reduced complement activation as compared toprotamine and similar activation to saline.

Example 18 Platelet Activation

The present example examines platelet activation in the presence ofheparin binding polymers. Platelet activation leads to aggregation,which can cause adverse effects such as thrombotic complications orarterial embolization. To measure platelet activation, blood wascollected in sodium citrate anticoagulant and the platelet rich plasma(PRP) isolated by centrifugation. Plasma was then incubated at 37° C.with JNK-1 at 10 mg/mL (9:1 dilution), polyethyleneimine (PEI) at 1mg/ml, insulin (a potent complement activator) as a positive control,saline, or PRP incubated with adenosine diphosphate and thrombin as apositive control for 30 minutes. Aliquots of the incubation wereassessed for activation state of the platelets using fluorescence flowcytometry. Expression of the platelet activation marker CD62P wasdetected using a Coulter Epics-XL (Miami, Fla.). Briefly, thepost-incubation polymer/platelet mix was diluted and incubated with themonoclonal anti-CD62P-FITC antibody. Data is reported as the percentageof platelets positive (activated) for CD62.

FIG. 10B demonstrates the platelet activation upon interaction withJNK-1 polymers and controls. Results show that JNK-1 does not induceplatelet activation as compared to known activators (PEI and positivecontrol). JNK-1 polymers induced activation that was no greater thanthat seen with saline alone; thus, the low level CD62 expression seenfor JNK-1 polymers can be attributed to the choice of suspending mediumfor the polymer.

Example 19 Blood Coagulation

The present example examines blood coagulation in the presence of aheparin binding polymer. JNK-1 polymers were tested for bloodcompatibility using the activated partial thromboplastin time (APTT) andthe prothrombin time (PT) in fresh human plasma. PT can be used toevaluate the extrinsic and common coagulation pathway and the resultsare expressed in seconds required for a fibrin clot to form after tissuethromboplastin (innovin) has been added to the blood sample. APTT isused to evaluate the intrinsic and common coagulation pathway. Theresults are expressed in seconds required for a fibrin clot to form inthe plasma sample after a partial thromboplastin reagent (actin) andcalcium chloride have been added to the sample. Blood was mixed with ameasured amount of citrate and the plasma was obtained bycentrifugation, using methods known in the art.

The effect of JNK-1 polymers on PT and APTT in platelet poor plasma isshown in FIG. 11A. A final JNK-1 concentration of 10 mg/ml is used at ina 9:1 dilution of plasma at 37° C. Control experiments were done addingidentical volumes of saline solution. JNK-1 polymer solutions wereincubated with platelet poor plasma for 5 minutes at 37° C. beforeadding PT and APTT reagents. The results show that JNK-1 polymers do notinfluence blood coagulation on their own as there was no significantdifference between the JNK-1 polymers and the saline control.

Plasma re-calcification time can be calculated as the time it takes todetect a clot after the addition of calcium ions. FIG. 11B shows theeffect of JNK-1 polymers on plasma re-calcification time in plateletpoor plasma. A final JNK-1 polymer concentration of 10 mg/ml was used ina 9:1 dilution of plasma at 37° C. Control experiments were done addingidentical volumes of saline solution. In this experiment no additionalagent is added to induce the coagulation except the calcium chloridesolution (recalcification). There was no significant difference betweenthe JNK-1 and plasma control. The results show that JNK-1 polymers donot influence the coagulation on their own.

Optical micrographs are shown in FIGS. 12A-12D of human red blood cellsafter one-hour incubation with JNK-1 polymers at differentconcentrations in whole blood (9:1 dilution) at 37° C. All images are at400× magnification. FIG. 12A is an incubation of JNK-1 polymers (10mg/ml). FIG. 12B is an incubation of JNK-1 polymers (1 mg/ml). FIG. 12Cis an incubation of polyethyleneimine (PEI) (1 mg/ml). FIG. 12D is anincubation of saline control. Results show that JNK-1 polymers do notinduce adverse effects such as red cell aggregation or hemolysis, unlikecationic polymers such as PEI (FIG. 12C).

Example 20 Heparin and Heparin Derivative Neutralization in Human Blood

Human plasma treated with sodium citrate was anti-coagulated withunfractionated heparin (UFH) or low molecular weight (LMWH) Tinzaparin.Samples subsequently mixed with JNK-1 and JNK-2 polymers showed completeneutralization for both UFH and Tinzaparin and did not showanticoagulation at high doses.

Conversely, for protamine treated samples, complete neutralizationoccurred at a higher dosage of protamine for UFH and not at all forTinzaparin. Furthermore, at high doses protamine had an anticoagulanteffect and was not an effective antidote.

These results were confirmed by analysis of the activated partialthromboplastin time (APTT), which showed that JNK-1 polymers werecapable of complete neutralization of UFH and LMWH but that protaminewas incapable of neutralizing LMWH and acted with less efficiency forUFH within a smaller dose window. Overall, JNK-1 polymers showed greaterUFH and LMWH neutralization capacity as compared to protamine incitrated plasma.

JNK-1 and JNK-2 polymers were also tested with human whole blood forefficacy in neutralizing UFH and LMWH. Thromboelastography measurementsshowed that human whole blood containing UFH or LMWH was efficientlyneutralized by the polymers JNK-1 and JNK-2. Conversely, protamine wasnot effective at neutralizing UFH or LMWH Tinzaparin and Enoxaparin interms of the time to clot formation in human whole blood. Althoughprotamine was able to allow UFH treated blood to regain the same clotstrength, it only capable of enabling LMWH Tinzaparin treated blood toregain the same clot strength at a lower dose and not at all at a highdose.

This study in human citrated plasma and whole blood indicates that theJNK-1 and JNK-2 polymers are effective for human samples with UFH andLMWH. Furthermore, these studies provide evidence that JNK-1 and JNK-2polymers display properties that are consistent with being useful forhuman clinical use.

The results in FIGS. 18A-18C demonstrate the neutralization of differentheparins: FIG. 18A: unfractionated heparin (UFH); FIG. 18B: LMWHTinzaparin; and FIG. 18C: LMWH Enoxaparin by the HBSPCM polymersdescribed in Table 2 (JNK-3 to JNK-8) in comparison to protamine inhuman blood in vitro.

Citrate plasma was anticoagulated with 2.0 U/ml UFH, Enoxaparin andTinzaparin and was treated with different concentrations of the HBSPCMpolymers and protamine (0.001 to 0.25 mg/ml). The APTT was measured foreach sample. Results show that the HBSPCM polymers, unlike protamine,effectively neutralize UFH and LMWH (Tinzaparin and Enoxaparin) and donot cause any anticoagulant effect at even higher concentration. HBSPCMpolymers can almost completely neutralize the anticoagulation of LMWH(Tinzaparin and Enoxaparin) at certain doses and showed greaterefficiency.

The results in FIG. 19 also demonstrate that HBSPCM polymers caneffectively neutralize LMWH using a thromboelastograph (TEG) in humanwhole blood. HBSPCM polymers JNK-5 and JNK-6 (0.1 mg/ml) were added tothe anticoagulated blood and compared to a control with no Enoxaparinadded (no anti-coagulation with Enoxaparin) and no neutralization(Enoxaparin control). The results demonstrate the ability of JNK-5 andJNK-6 to neutralize Enoxaparin in terms of the TEG strength. The TEGtrace of the enoxaparin neutralized blood is almost identical to salinecontrol, which was not coagulated. Together with FIG. 18, these datademonstrate the efficiency of HBSPCM polymers such as JNK-5 and JNK-6 toneutralize LMWH under in vitro conditions.

The data in FIGS. 24A-24C demonstrate the neutralization of differentheparins ((FIG. 24A): unfractionated heparin (UFH); (FIG. 24B): LMWHTinzaparin; and (FIG. 24C): LMWH Enoxaparin) by the HBSPCM polymersdescribed in Table 3 (JNK-9, JNK-10, JNK-11) in comparison to protaminein human plasma in vitro. Citrate plasma was anticoagulated with UFH(2.0 IU/mL), Enoxaparin (2.0 IU/mL) and Tinzaparin (1.14 IU/mL) and wastitrated with different concentrations of the HBSPCM polymers andprotamine (0.001 to 0.25 mg/ml). The APTT was measured for each sample.Results show that the HBSPCM polymers, unlike protamine, effectivelyneutralize UFH and LMWH (Tinzaparin and Enoxaparin) and do not cause anyanticoagulant effect at higher concentrations than protamine. The HBSPCMpolymers JNK-9, JNK-10 and JNK-11 can almost completely neutralize theanticoagulation of LMWH (Tinzaparin and Enoxaparin) at certain doses andshowed greater efficiency.

Example 21 High Binding Affinity of Heparin to HBSPCM Polymer JNK-2

The present example examines the binding affinity of a heparin bindingpolymer to heparin. The HBSPCM polymer JNK-2 was tested for UFH, LMWHand fondaprinux binding by isothermal titration calorimetry. Isothermaltitration calorimetry (ITC) was performed using a VP-ITC (MicroCal,Inc., Northampton Mass.). Samples were in PBS buffer pH 7.2. Titrationswere performed by injecting consecutive 5-10 μL aliquots of JNK-2solution into the ITC cell (volume=1.4 mL) containing heparin or heparinderivatives. The ITC data were corrected for the heat of dilution of thetitrant by subtracting mixing enthalpies for 5-10 μL injections of JNK-2solution into heparin- and heparin derivative-free buffer. At least twoindependent titration experiments were performed for each system at 25°C. to determine the binding constant of HBSPCM to heparin derivatives.Binding stoichiometry, N, enthalpy, ΔH_(b), entropy, ΔS, and equilibriumassociation constants, K_(a), were determined by fitting the correcteddata to a bimolecular interaction model. This study demonstrated highbinding affinity and enthalpy of interaction of JNK-2 molecules toheparin derivatives. Table 5 shows the summary of isothermal titrationcalorimetry (ITC) results for JNK-2 titrations with unfractionatedheparin, Enoxoparin and Fondaparinux.

TABLE 5 SUMMARY OF JNK-2 TITRATIONS Macromolecule N¹ K_(a) (M^(−I)) ΔG(kcal/mol) ΔH (kcal/mol) TΔS (kcal/mol) Unfractionated heparin 0.9(±0.I)  1A (±0.7) × 106 −8.3 (±0.2)  −94 (±15)  −85 (±15) LMWH(Enoxoparin) 2.95 (±0.08) 1.3 (±0.2) × 105 −7.0 (±0.I) −34.8 (±0.5)−27.8 (±0A) Fondaparinux  2.3 (±0.0I) 1.5 (±0.2) × 105 −7.1 (±0.I) −17.9(±0.2) −10.9 (±0.I) ¹N = number of heparin molecules that binds to oneJNK-2 (molecular weight = 48 kDa). Experiments performed at 25° C.(errors represent the standard deviations of replicate experiments). ~G,~H, TΔS are calculated per mole of macromolecule.

Example 22 Heparin and Heparin Derivative Neutralization in Living Rats

The present example examines heparin binding polymer effectiveness invivo. HBSPCM polymers were tested for their ability to neutralize UFHand LMWH in living rats. Rats were first injected with heparin orheparin derivative, and then injected with the JNK-1 polymers, protamineor saline control to neutralize the previously administered heparin orheparin derivative. Neutralization was measured by detecting thepercentage of factor Xa using an anti-Xa assay as a measure ofanticoagulant activity.

The results are shown in FIGS. 8A-8D. The data in FIG. 8A-8Ddemonstrates the ability of JNK-1 and JNK-5 (HBSPCM-3) polymers toneutralize UFH and LMWH (Tinzaparin and Enoxaparin) in rats. FIG. 8Ashows data for protamine+UFH and UFH alone and FIG. 8B shows data forprotamine+Tinzaparin and Tinzaparin alone, were compared to JNK-1polymers+UFH (FIG. 8A) and JNK-1 polymers+Tinzaparin (FIG. 8B),respectively. In addition, Enoxaparin alone and JNK-1polymers+Enoxaparin were compared (FIG. 8C). An anti-Xa assay was usedto measure heparin activity in rat blood. In this pilot study, rats wereinjected with 25 U of UFH, Tinzaparin or Enoxaparin (0 minutes). Bloodwas collected after UFH, Tinzaparin, or Enoxaparin infusion (5 minutes).Approximately 1 mg of JNK-1 polymers or protamine (dose was 3 mg/kg) wasinjected at 7 minutes and the blood was collected at 12 minutes afterUFH/Tinzaparin/Enoxaparin infusion (5 minutes after JNK-1 infusion), and17 minutes (10 minutes after JNK-1 polymer infusion). For subjectsreceiving Enoxaparin, blood was collected at additional time points (25minutes and 30 minutes). Blood samples collected before the UFH,Enoxaparin or Tinzaparin infusion were used as a base control. JNK-1polymers fully neutralize the anticoagulant activity of UFH, Enoxaparinand Tinzaparin and factor Xa levels reach almost to control levels afterJNK-1 polymer infusion. Each data point shown reflects an average andstandard deviation from five rats. The chronological order of eventsduring the experiment is given on the bottom portion of the graphs inFIGS. 8A-8C; arrows indicate the time points.

The data in FIG. 8A show that for unfractionated heparin (UFH), theJNK-1 polymers can completely neutralize the UFH within at most fiveminutes after infusion, performed equally well as protamine andoutperformed the control. The data in FIG. 8B shows that for lowmolecular weight heparin (LMWH), the JNK-1 polymers can completelyneutralize the LMWH within at most five minutes after infusion,outperforming both protamine and the control, which could not completelyneutralize the LMWH.

The data in FIG. 8D shows the ability of JNK-5 polymer to neutralizeLMWH enoxaparin in rats. Rats were injected with 25 U of Enoxaparin (0minutes). Blood was collected after enoxaparin infusion (5 minutes).Approximately 1 mg of JNK-5 polymers or protamine (dose was 3 mg/kg) wasinjected at 7 minutes and the blood was collected at 12 minutes afterenoxaparin infusion (5 minutes after JNK-5 infusion), 17 minutes (10minutes after JNK-5 polymer infusion) and 65 minutes (one hour afterJNK-5 polymer infusion). For subjects receiving enoxaparin, blood wascollected at additional time point (25 minutes). Blood samples collectedbefore the enoxaparin infusion were used as a base control. JNK-5polymers fully neutralize the anticoagulant activity of enoxaparin andfactor Xa levels reach almost to control levels after JNK-5 polymerinfusion. Each data point shown reflects an average and standarddeviation from five rats. The chronological order of events during theexperiment is given on the bottom portion of the graphs in FIG. 8D;arrows indicate the time points.

Example 23 Heparin and Heparin Derivative Neutralization in Living Rats

The data in FIGS. 21A and 21B demonstrate the ability of anotherembodiment, JNK-3 polymers, to neutralize UFH and LMWH Enoxaparin inliving rats. Unfractionated heparin (UFH) alone and JNK-3 polymers+UFHwere compared (FIG. 21A). Enoxaparin alone and JNK-3 polymers+Enoxaparinwere compared (FIG. 21B). An anti-Xa assay was used to measure heparinactivity in rat blood.

In this study, rats were injected with 25 U of UFH or Enoxaparin (0minutes). Blood was collected after UFH or Enoxaparin infusion (3minutes). 1 mg of JNK-3 polymers was injected at 5 minutes and the bloodwas collected at 7, 10, 15 and 35 minutes after UFH/Enoxaparin infusion.For subjects receiving UFH infusion, blood was also collected at 65minutes. Blood samples collected before the UFH or Enoxaparin infusionwere used as a base control. JNK-3 polymers fully neutralize theanticoagulant activity of UFH and Enoxaparin and factor Xa levels reachalmost to control levels. The chronological order of event during theexperiment is given on the bottom portion of the graphs in FIG. 21;arrows indicate the time points.

These in vivo studies indicate that heparin binding polymers, includingboth JNK-1 and JNK-3 polymers, work effectively on both UFH and LMWH inthe intact living system of rat model, providing evidence that thefindings may be extrapolated to systemic human use.

Example 24 Heparin and Heparin Derivative Neutralization in anExtracorporeal Device

The present example is directed to an extracorporeal device that can beused to remove and/or reduce heparin and derivatives thereof. The bloodfrom a subject previously administered with heparin or heparinderivatives is directed into an extracorporeal circuit bypass of acirculation system is filtered for the removal of heparin or heparinderivatives for reuse by the subject.

The extracorporeal device can include a structure including, but notlimited to, a cylindrical cartridge, hollow tubing, a rigid or flexiblevesicle, and the like. HBSPCM polymers are attached to some the part ofthe extracorporeal device coming into contact with blood and areattached either directly, or through a means of tethering. Tethering caninclude, but is not limited to, polymer brushes or surfacefunctionalities or by non-covalent complexation (electrostatic).

As blood is moved through the extracorporeal device, after it leaves thesubject and before it is returned to the subject, HBSPCM polymersattached to the device neutralize heparin or heparin derivatives in theblood.

Example 25 Blood Compatibility Analysis

Blood compatibility of the polymers was assessed by several methods(blood coagulation, platelet activation, complement activation, red cellhemolysis and aggregation. The change in activated partialthromboplastin time (APTT) was evaluated in comparison to a salinecontrol. A significant change in the APTT shown by the heparin bindingpolymers as compared to a saline control suggests that the HBSPCMpolymers are not interfering with blood coagulation proteins such astissue factor and other coagulation factors.

FIG. 15 shows an analysis of blood compatibility by demonstrating theactivated partial thromboplastin time (APTT) assay by the HBSPCMpolymers described in Table 2. Blood coagulation is measured using APTT.The amine content changed the APTT and protamine resulted in a differentblood coagulation profile.

FIG. 16 shows another analysis of blood compatibility usingthromboelastograph (TEG) in terms of coagulation time (R) and kineticsparameter (K) at a polymer concentration of 0.5 mg/ml for the polymersdescribed in Table 2. TEG studies yielded the cumulative effect ofseveral components of coagulation (global homeostasis) as a function oftime. The polymers with higher amine content and protamine resulted in adifferent blood coagulation profile.

Example 26 Rate of Red Blood Cell Lysis

This example examines the rate of red blood cell lysis in the presenceof a heparin binding polymer.

Red blood aggregation in plasma/buffer is not easily disrupted by shearforces and can result in high blood viscosity and complications. Severeaggregation can cause cell damage and hemolysis, leading to a variety ofcomplications including anemia and jaundice. Red blood cell hemolysisshows the toxicity of the compounds tested.

In the present example, the degree of hemolysis was observed bymonitoring the degree of red color present in the supernatant plasma orbuffer upon centrifugation of the red blood cells after incubation for 1hour at 37° C. with heparin binding polymers. Red blood cell lysis wasdetermined by measuring the amount of extracellular hemoglobin againstthe total hemoglobin concentration. Briefly, 50 microliters of red bloodcell suspension and 100 microliters of red blood cell supernatant weremixed with Drabkin's reagent (1 ml each) and the absorbance wasmonitored at 540 nm running Drabkin's reagent as blank.

FIG. 17A and FIG. 17B demonstrate the results of a red blood cell lysisassay, which measured the percent of red blood cells that were lysedafter incubating different HBSPCM polymers as described in Table 2 withred blood cells at 37° C. for 1 hour at two different concentrations:(FIG. 17A) 0.1 mg/ml; (FIG. 17B) 5 mg/ml. Shown on the left side of FIG.17A and FIG. 17B are photographs of red blood cell suspensionscentrifuged after incubation with the polymers.

Example 27 Tolerance of HBSPCM Polymers in Mice

The present example examines the degree of tolerance that animals haveto various heparin binding polymers, and to HBSPCMs in particular.

FIG. 22 shows the body weights over time (0 to 29 days post injection)of female BALB/c mice treated with JNK-3 polymers (23 kDa) by bolusintravenous injection of increasing concentration of JNK-3 polymers (1mg/kg to 50 mg/kg). Mice were randomly assigned to dosing groups. Allinjections (200 μL of HBSPCM in saline) were performed as scheduled withno adverse reactions noted. No significant decreases in body weight orbehavioral changes were noted following administration of JNK-3polymers. All the mice were terminated as per study protocol on the29^(th) day with no notation on necropsy.

The results show that JNK-3 polymers can be safely dosed by bolusintravenous administration in female BALB/c mice at a dose of up to 50mg/kg (maximum dosage given). The tolerated dose is at least 16-foldhigher as compared to protamine, which has a maximum tolerated dose of 3mg/kg. The study was carried out in the Department of AdvancedTherapeutics, British Columbia Cancer Agency, Vancouver, under anapproved UBC animal ethics protocol.

Example 28 Cytotoxicity of HBSPCM Polymers

The present example examines the cytotoxicity of a heparin bindingpolymer. The lactate dehydrogenase (LDH) assay demonstrates thecytotoxicity present in a sample containing cells based on the activityof LDH released from damaged cells. The LDH assay was used to assess thecytotoxicity of a HBSPCM polymer, JNK-3.

FIG. 23 demonstrates the LDH activity in serum of female BALB/c micetreated with JNK-3 polymers (23 kDa) after an intravenously appliedbolus given on day 0 of increasing concentration (1 mg/kg to 50 mg/kg)as measured on the termination day (29^(th) day of post injection). Theresults demonstrate that the LDH activity associated with all doses waseither below or not dramatically greater than the control activitylevel. Results dramatically higher than the control activity level wouldhave been indicative of liver and cell toxicity associated with theHBSPCM polymers (JNK-3). Base LDH activity levels for control mice werearound 100 IU/I (dotted line in FIG. 23).

Example 29

The present example outlines a method of making HBSPCM polymers JNK-1and JNK-2. The HBSPCM polymers JNK-1 and JNK-2 were synthesized usingsimilar protocols to that of polymers JNK-3 to JNK-11, The averagenumber of charge in JNK-1 and JNK-2 are 276 and 112 respectively.Synthetic procedure for JNK-1 is given below.

Synthesis of Polymer JNK-1 (116 kDa HBSPCM Polymer)

The synthesis steps include the following, Firstly, a HPG-PEG-116Kprecursor polymer was synthesized using the following steps. Athree-necked round bottomed flask was cooled under vacuum and filledwith argon. To this, 1,1,1-Tris(hydroxymethyl)propane (TMP, 0,120 g) andpotassium methylate (25 wt % solution in methanol, 0.110 mL) were addedand stirred for 30 minutes Methanol was removed under vacuum for 4hours. The flask was heated to 95° C. and glycidol (6 mL) was added overa period of 15 hours. After complete addition of monomer, the reactionmixture was stirred for additional 3 hours. MPEG-epoxide-400 (20 mL) wasadded over a period of 12 hours. The reaction mixture was stirred foradditional 4 hours. The polymer was dissolved in methanol, passedthrough Amberlite IRC-50 resin to remove the potassium ions and twiceprecipitated from diethyl ether. The polymer was dissolved in water anddialyzed against water using MWCO-1000 membrane for 3 days with periodicchanges in water.

The JNK-1 polymer was synthesized from the HPG-PEG-116K precursorpolymer described in the previous paragraph as follows, HPG-PEG-116K (16g) was dissolved in 100 mL of pyridine. To this, p-toluene sulfonylchloride was added and stirred at room temperature for 24 hours.Pyridine was removed by rotary evaporation; the polymer was dissolved in0.1 N HCl and dialyzed overnight. The polymer was isolated by freezedrying. The dried polymer and tris(2-aminoethylamine) (2 mL) weredissolved in 1,4-dioxane (100 mL) and refluxed for 24 hours. Dioxane wasremoved under vacuum. The polymer was dissolved in minimum amount ofmethanol and precipitated twice from diethyl ether. Polymer wasdissolved in water and dialyzed against water using MWCO-1000 membranefor 2 days. The resulting polymer solution was added to a mixture offormaldehyde (15 mL) and formic acid (15 mL) at 0° C. The reactionmixture was refluxed overnight. After cooling to room temperature, thepH of the solution was adjusted to 10 using NaOH and the polymer wasextracted with dichloromethane. Dichloromethane was removed undervacuum; the polymer dissolved in water and dialyzed using MWCO-1000membrane for 3 days. The yield of JNK-1 polymer was 10 g, and the aminecontent (by conductometric titration) was 5.4 mol %.

Synthesis of JNK-2 Polymer (48 kDa HBSPCM Polymer)

JNK-2 polymer was synthesized using similar procedure as that for JNK-1.First, a HPG-PEG-48K precursor polymer was synthesized and a portion ofthe hydroxyl groups were converted into tertiary amino groups by asimilar procedure. Amine content of the polymer (by conductometrictitration) was 7.6 mol %.

Example 30 Comparative Analysis of UFH Embodiments Vs. Tinzaparin andEnoxaparin

This example was performed to present a comparative analysis of UFHembodiments vs. Tinzaparin and Enoxaparin.

FIGS. 24A-24C show the ability of HBSPCM polymers JNK-9, JNK-10 andJNK-11 to neutralize UFH and LMWH tinzaparin and enoxaparin incomparison to protamine and saline control as measured by activatedpartial thromboplastin time (APTT).

FIG. 24A shows the neutralization of UFH by JNK-9, JNK-10 and JNK-11.

FIG. 24B shows the neutralization of LMWH Tinzaparin by JNK-9, JNK-10and JNK-11.

FIG. 24C shows the neutralization of LMWH Enoxaparin by JNK-9, JNK-10and JNK-11.

Ability of HBSPCM polymers JNK-9, JNK-10 and JNK-11 was studied for therange of concentration from 0.01 to 0.25 mg/mL prepared in 0.15 Msaline. Citrated blood was spun at 3000 RPM for 10 min to obtainplatelet poor plasma (PPP). The PPP was then heparinized by adding 45 μLof heparin (UFH, LMWH etc.) to 2200 μL of PPP—final concentration of 2.0IU/mL for UFH and Tinzaparin and 1.14 IU/mL for Enoxaparin. Normalcontrol was prepared by adding 25 μL of 0.15 M saline to 250 μL of PPP.25 μL of each antidote solution (polymer or protamine) was added to 225μL of heparinized PPP. 250 μL of APTT reagent (actin FSL) was then addedand samples were incubated for 3 min at 37° C. 250 μL of normal controlwas combined with 250 μL of APTT reagent and also incubated for 3 min.50 μL of 0.025M CaCl₂ was added to 100 μL aliquots of normal control andsample (in triplicate). A heparinized control containing 225 μL of PPPand 25 μL of saline was also prepared and run similar to normal control.The results indicate that HBSPCM polymers JNK-9, JNK-10 and JNK-11effectively neutralize UFH and LMWH tinzaparin and enoxaparin over abroader range of concentration, while protamine neutralizes UFH and LMWHonly in the narrow concentration range, and at higher concentrationsprotamine possesses anticoagulant activity.

Example 31 Blood Compatibility of Various HBSPCMs

This example examined blood compatibility of various HBSPCMs, and inparticular the effect of the charge on the polymer.

FIG. 25 shows the complement activation of HBSPCM polymer having amolecular weight of 23 kDa with different charge density (e.g., numberof amine groups). The level of complement activation was measured by aCH50 sheep erythrocyte lysis assay. Ten microliter of each of the 0.5and 5 mg/mL polymers or protamine was mixed with 90 μL of serum for 1 hat 37° C. The final concentration of polymer/protamine to serum was 0.05and 0.5 mg/mL. One mg/mL heat-activated human IgG and 5 mM EDTA, aftermixing with serum, were the positive and negative controls,respectively, for the study. 60 μL of post-incubation serum/polymer orprotamine mixture was diluted by 120 μL of GVB²⁺ (CompTech).Seventy-five microliter of GVB²⁺diluted serum/polymer or protaminemixture was incubated for 1 h at 37° C. with 75 μL of Ab-sensitizedsheep erythrocyte (CompTech). The reaction was stopped by addition of300 μL cold GVB-EDTA to each sample. The samples were centrifuged andthe optical density of supernatant was measured at 540 nm One hundredpercent lysis of the sheep erythrocyte was done by dH₂O. The percentageof complement activation was calculated by 100-% lysis of sheeperythrocytes.

The results reveal that 23 kDa HBSPCM polymer with 16-96 charges perpolymer molecular do not activate the complement system, whereas thepolymer control sample with 92 charges (where the primary amino groupsare not methylated) and protamine showed significant complementactivation.

FIG. 26 shows the platelet activation of 23 kDa HBSPCM polymers having adifferent number of amine groups per polymer molecule (16-80 aminegroups/polymer molecule). The level of platelet activation wasquantified by flow cytometry. Ninety microliter of PRP was incubated at37° C. with 10 μL of 5 mg/mL of polymer or protamine (finalconcentration 0.5 mg/mL). After 1 h, aliquots of the incubation mixtureswere removed for assessment of the platelet activation state. Fivemicroliter of post-incubation platelet/polymer or protamine mixture,diluted in HEPES buffer, was incubated for 20 minutes in the dark with 5μL of monoclonal anti-CD62-PE (Immunotech). The samples were thenstopped with 0.5 mL of phosphate-buffered saline solution. The level ofplatelet activation was analyzed in a BD FACSCanto II flow cytometer(Becton Dickinson) by gating platelets specific events based on theirFITC-CD42 fluorescence and light scattering profile. Activation ofplatelets was expressed as the percentage of platelet activation markerCD62-PE fluorescence detected in the 10,000 total events counted.Duplicate measurements were done, the mean of which was reported.Controls were done for the flow cytometric analysis. One U/ml of bovinethrombin (Sigma) was used as a positive control, and PE conjugated goatanti-mouse IgG polyclonal antibodies (Immunotech) was used as thenon-specific binding control.

The results indicate that 23 kDa HBSPCM polymers with 16-80 number ofcharges per polymer molecule do not activate platelets, whereasprotamine showed significant levels of platelet activation.

Example 32 Tolerance of HBSPCM Vs. Protamine

This example examined the tolerance of HBSPCM vs. protamine in mice.

FIGS. 27 and 28 show the single dose tolerability of HBSPCM polymerJNK-1 and protamine respectively, compared to a vehicle control (saline)in female Balb/cJ mice.

For the study, the mice were individually weighed and injected with aprescribed dose (mg/Kg) based on their individual weights (200 μL/20 g)of JNK-1 (FIG. 27), protamine (FIG. 28) and control saline. Mice werecontinually monitored for acute signs of toxicity for the first twohours following administration and for additional toxicities 28 dayspost administration. Body weights of the mice were measured over aperiod of 29 days.

The results are shown in FIG. 27 (HBSPCM) and FIG. 28 (Protamine). As isclearly shown in FIG. 27, these heparin binding polymers are not toxicin mice up to the maximal dose studied (200 mg/Kg). In contrast, asshown in FIG. 28, the maximum dose of protamine tolerated in mice was 20mg/kg, with mice dying at doses above 20 mg/kg.

Example 33 Biodistribution of HBSPCM-3

The biodistribution of HBSPCM-3 (23 kDa) was studied. The molecule wasradio-labeled (tritium) and was intravenously injected into BALB/c mice.Radio-activity in different organs and plasma was measured.

FIG. 29 and FIG. 30 show the results of the study conducted to determinethe pharmacokinetics and biodistribution of HBSPCM-3 (23 kDa) polymer infemale Balb/cJ mice following bolus intravenous injection.

32 female Balb/cJ mice were administered with HBSPCM-3 polymer at a doseof 20 mg/Kg. Blood was collected at 5 min, 30 min, 1 h, 2 h, 4 h, 8 h,24 h and 48 h post intravenous single bolus injection. Four mice wereterminated at each time point by CO₂ inhalation and blood was collectedby cardiac puncture. 100 μL whole blood sample was placed in pre-weighedtubes and the remaining volume was processed for plasma. Upontermination, the liver, spleen, lung, kidney and heart were weighed andprocessed for scintillation counting. Livers were further processed byadding water to make a 20% homogenate solution.

As shown in the charts, there was no accumulation in the major organssuch as liver, kidney and spleen suggesting non-toxic behavior ofHBSPCM-3.

Example 34 Biodistribution of HBSPCM-1

The biodistribution of HBSPCM-1 (116 k) was studied. The molecule wasradio-labeled (tritium) and was intravenously injected into BALB/c mice.Radio-activity in different organs and plasma was measured. FIG. 31 andFIG. 32 show the results of a study conducted to determine thepharmacokinetics and biodistribution of HBSPCM-1 (116 kDa) polymer infemale Balb/cJ mice following bolus intravenous injection.

36 female Balb/cJ mice were administered with HBSPCM-1 polymer at a doseof 20 mg/kg. Blood was collected at 5 min, 30 min, 1 h, 2 h, 4 h, 8 h,24 h, 48 h, and 72 h post intravenous single bolus injection. Four micewere terminated at each time point by CO₂ inhalation and blood wascollected by cardiac puncture. 100 μL whole blood sample was placed inpre-weighed tubes and the remaining volume was processed for plasma.Upon termination, the liver, spleen, lung, kidney and heart were weighedand processed for scintillation counting. Livers were further processedby adding water to make a 20% homogenate solution.

The results are presented in FIGS. 31A, 31B, 31C, 31D, 32A, and 32B. Asshown in the charts, there was no accumulation in the liver and spleensuggesting non-toxic behavior of HBSPCM.

Example 35 HBP to Reduce the Level of Effective Heparin

The present example outlines how a heparin binding polymer can be usedto reduce the level of effective heparin in a subject.

A subject receiving treatment for a burn is administered an amount ofheparin. The amount of heparin is deemed to be excessive at some pointand the subject is then administered an adequate amount of HBSPCM-3, tocounteract the amount excessive amount of heparin.

As will be appreciated by those of skill in the art, in light of thepresent disclosure, in some embodiments, the polymer-bound heparin canbe cleared from the body depending on the molecular weight. In someembodiments, polymers of molecular weight less than 50 kDa can becleared through the kidney, while the higher molecular weight polymerscan be excreted through feces.

Example 36 Heparin Binding Polymer for Reducing Heparin Levels for aSubject Ex Vivo

The present example outlines how a heparin binding polymer can be usedto reduce heparin levels for a subject ex vivo.

A subject receiving treatment for acute coronary syndrome isadministered an amount of heparin. At some point, it is desired toreduce the amount of heparin in the subject, and thus, blood is takenfrom the subject and run through a filtering device that includesHBSPCM-1 immobilized on a support such that the blood passes over theHBSPCM-1. This allows the polymer to bind to and remove the heparin,thereby providing filtered blood. The filtered blood is then returned tothe subject.

Example 37 Heparin Binding Polymer for Heparin Isolation

The present example outlines how a heparin binding polymer can be usedto isolate heparin.

A solution containing heparin is provided. The solution can be theresulting mixture from a process of heparin preparation. A heparinbinding polymer, which includes a polyglycerol core and polyvalentcationic groups, is immobilized on a surface. The solution is flowedacross the surface at room temperature and the heparin is allowed tobind to the heparin binding polymer. The surface is then washed withwater (in the alternative, a buffer solution (0.15 M NaCl or PBS orother similar solutions) can be used) and then the isolated heparin canbe eluted from the polymer with a salt solution (1 to 3 M saltsolution), which can be performed at room temperature, or alternatively,at body temperature. Some of the salt can then be removed from thesolution to provide an isolated heparin composition in an osmoticallyphysiological salt solution.

Example 38 HBSPCM Polymers for Drug Delivery Applications

The present example outlines the use of HBSPCM polymers for the deliveryof anionic drugs such as methotrexate, phenoxymethyl penicillin,insulin, indomethacin, diclofenac etc. The cationic nature of HBSPCMpolymers results in effective binding to these anionic drug moleculeswhich could be released at a controlled rate once inside the body.

Other aspects and features various embodiments will become apparent tothose ordinarily skilled in the art upon review of the followingdescription of specific examples.

The present disclosure is not to be limited in terms of the particularembodiments described in this application, which are intended asillustrations of various aspects. Many modifications and variations canbe made without departing from its spirit and scope, as will be apparentto those skilled in the art. Functionally equivalent methods andapparatuses within the scope of the disclosure, in addition to thoseenumerated herein, will be apparent to those skilled in the art from theforegoing descriptions. Such modifications and variations are intendedto fall within the scope of the appended claims. The present disclosureis to be limited only by the terms of the appended claims, along withthe full scope of equivalents to which such claims are entitled. It isto be understood that this disclosure is not limited to particularmethods, reagents, compounds, compositions or biological systems, whichcan, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting.

With respect to the use of substantially any plural and/or singularterms herein, those having skill in the art can translate from theplural to the singular and/or from the singular to the plural as isappropriate to the context and/or application. The varioussingular/plural permutations may be expressly set forth herein for sakeof clarity.

It will be understood by those within the art that, in general, termsused herein, and especially in the appended claims (e.g., bodies of theappended claims) are generally intended as “open” terms (e.g., the term“including” should be interpreted as “including but not limited to,” theterm “having” should be interpreted as “having at least,” the term“includes” should be interpreted as “includes but is not limited to,”etc.). It will be further understood by those within the art that if aspecific number of an introduced claim recitation is intended, such anintent will be explicitly recited in the claim, and in the absence ofsuch recitation no such intent is present. For example, as an aid tounderstanding, the following appended claims may contain usage of theintroductory phrases “at least one” and “one or more” to introduce claimrecitations. However, the use of such phrases should not be construed toimply that the introduction of a claim recitation by the indefinitearticles “a” or “an” limits any particular claim containing suchintroduced claim recitation to embodiments containing only one suchrecitation, even when the same claim includes the introductory phrases“one or more” or “at least one” and indefinite articles such as “a” or“an” (e.g., “a” and/or “an” should be interpreted to mean “at least one”or “one or more”); the same holds true for the use of definite articlesused to introduce claim recitations. In addition, even if a specificnumber of an introduced claim recitation is explicitly recited, thoseskilled in the art will recognize that such recitation should beinterpreted to mean at least the recited number (e.g., the barerecitation of “two recitations,” without other modifiers, means at leasttwo recitations, or two or more recitations). Furthermore, in thoseinstances where a convention analogous to “at least one of A, B, and C,etc.” is used, in general such a construction is intended in the senseone having skill in the art would understand the convention (e.g., “asystem having at least one of A, B, and C” would include but not belimited to systems that have A alone, B alone, C alone, A and Btogether, A and C together, B and C together, and/or A, B, and Ctogether, etc.). In those instances where a convention analogous to “atleast one of A, B, or C, etc.” is used, in general such a constructionis intended in the sense one having skill in the art would understandthe convention (e.g., “a system having at least one of A, B, or C” wouldinclude but not be limited to systems that have A alone, B alone, Calone, A and B together, A and C together, B and C together, and/or A,B, and C together, etc.). It will be further understood by those withinthe art that virtually any disjunctive word and/or phrase presenting twoor more alternative terms, whether in the description, claims, ordrawings, should be understood to contemplate the possibilities ofincluding one of the terms, either of the terms, or both terms. Forexample, the phrase “A or B” will be understood to include thepossibilities of “A” or “B” or “A and B.”

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, such as in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” and the like include the number recited andrefer to ranges which can be subsequently broken down into subranges asdiscussed above. Finally, as will be understood by one skilled in theart, a range includes each individual member. Thus, for example, a grouphaving 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, agroup having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells,and so forth.

From the foregoing, it will be appreciated that various embodiments ofthe present disclosure have been described herein for purposes ofillustration, and that various modifications may be made withoutdeparting from the scope and spirit of the present disclosure.Accordingly, the various embodiments disclosed herein are not intendedto be limiting, with the true scope and spirit being indicated by thefollowing claims.

The invention claimed is:
 1. A method of processing a subject's blood, the method comprising: providing a heparin binding device, the device comprising: a support; and a heparin binding polymer immobilized on the support, wherein the heparin binding polymer comprises: a first dendritic polyol; and one or more cationic moieties attached to the first dendritic polyol, wherein the heparin binding polymer comprises at least one of: a) the structure of Formula (I):

wherein L₁ is the first dendritic polyol, L₂ is a second dendritic polyol, L₃ is a third dendritic polyol, L₄ is a fourth dendritic polyol, and L₅ is a fifth dendritic polyol, or b) the structure of Formula (II):

wherein L₁ is the first dendritic polyol and L₂ is a second dendritic polyol, and wherein L₃ is a third dendritic polyol, and L₄ is a fourth dendritic polyol; withdrawing blood from a subject; contacting the blood to the heparin binding polymer; and returning at least part of the blood to the subject, thereby processing the subject's blood.
 2. The method of claim 1, wherein the subject's blood contains heparin.
 3. The method of claim 2, wherein the heparin is exogenous heparin.
 4. The method of claim 3, wherein the exogenous heparin binds to the heparin binding polymer and is thereby at least partially removed from the subject's blood.
 5. The method of claim 3, further comprising the step of selecting a heparin binding polymer based upon the type of exogenous heparin administered to the subject.
 6. The method of claim 5, wherein a heparin binding polymer of a particular molecular weight is selected based upon the type of exogenous heparin.
 7. The method of claim 5, wherein a heparin binding polymer comprising a particular number of cationic groups is selected based upon the type of exogenous heparin.
 8. The method of claim 5, wherein the heparin binding polymer works for at least one of: unfractionated heparin (UFH), low molecular weight heparin (LMWH), ultra low molecular weight heparin (ULMWH), fondaparinux, idraparinux enoxaparin, dalteparin, tinzaparin, semuloparin, or heparinoid.
 9. The method of claim 4, wherein returning at least part of the blood to the subject further comprises fortifying the blood with one or more blood constituents that also bind to the heparin binding protein, wherein the fortifying is performed after the blood has contacted the heparin binding polymer. 